Methods for evaluating tumor cell spheroids using 3d microfluidic cell culture device

ABSTRACT

Provided herein are methods for evaluating tumor cell spheroids in a three-dimensional microfluidic device by determining changes in the relative levels of live cells and dead cells in aliquots cultured under different conditions. Methods described herein allow ex vivo recapitulation of the tumor microenvironment such that the in vivo effectiveness of a test compound in treating tumor tissue may be predicted.

RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US2018/025390, filed Mar. 30, 2018, and entitled “METHODS FOREVALUATING TUMOR CELL SPHEROIDS USING 3D MICROFLUIDIC CELL CULTUREDEVICE,” which claims the benefit of U.S. provisional application No.62/480,192, filed on Mar. 31, 2017, the entire contents of which isincorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under K08 CA138918-01A1and R01 CA190394-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Existing patient-derived cancer models, including circulating tumorcells (CTCs), organoid cultures, and patient-derived xenografts (PDXs)can guide precision cancer therapy, but take weeks to months to generateand lack the native tumor immune microenvironment. Current approaches tostudy anti-tumor immune responses in patients are also limited by remotemeasurements in whole blood or plasma, or static assessment of biopsies.

Recently there has been developed a 3D microfluidic device for theshort-term culture of murine- and patient-derived organotypic tumorspheroids. Cultured tumor spheroids are believed by some to more closelyresemble the native immune microenvironment.

It would be desirable to be able to assess the effects of single drugsand combinations of drugs on tumor cells in the tumor micro-environment.It would be particularly helpful if there were a way to evaluatesimultaneously the effects of a combination of immune blockade andanticancer compounds ex vivo. To date, efforts to evaluate tumor cellshave been confounded by (i) the tumor cells changing when removed fromthe body, (ii) the absence of an intact immune system communicating withthe tumor microenvironment when the tumor cells are removed from thebody, (iii) the inability to distinguish the effects of tumor removaland culturing from the effects of a drug applied in vitro, and (iv) thecomplexity of measuring at a molecular level immune based markers andother markers which might predict whether a drug might work in vivobased on experiments conducted in vitro. To date, there is no simpleassay for evaluating in in a high throughput manner the effects of drugson human cancer cells in a native tumor microenvironment.

SUMMARY

It has been discovered, surprisingly, that the tumor microenvironment ofa cultured tumor spheroid contains sufficient immune components to bepredictive of a drug's activity when administered in vivo. It has beendiscovered, surprisingly, that the tumor microenvironment of a culturedtumor spheroid contains sufficient immune components such thatcombinations of immune blockade compounds and anti-cancer drugs can beassessed, the results being predictive of administering the drugs invivo. It has been discovered, surprisingly, that the tumormicroenvironment of a cultured tumor spheroid can be evaluated opticallyand reproducibly, to establish the effects of drugs on the tumor cellswithin the spheroids, while distinguishing the effects on tumor cellsresulting from the act of culturing the tumor cells. It has beendiscovered, surprisingly, that such techniques for evaluating tumorspheroids can be utilized in a high throughput method for determiningthe relative amounts of live and dead cells in an environment mimickingthat occurring in vivo.

In one aspect, the invention involves a novel approach for evaluating exvivo response to Immune Checkpoint Blockade (ICB) using murine- andpatient-derived organotypic tumor spheroids (MDOTS/PDOTS) cultured in a3-dimensional microfluidic system. Spheroids isolated from fresh mouseand human tumor samples retain autologous lymphoid and myeloid cellpopulations, including antigen-experienced tumor infiltrating CD4 andCD8 T lymphocytes, and respond to ICB in short-term ex vivo culture.Tumor killing was recapitulated ex vivo using MDOTS derived from theanti-PD-1 sensitive MC38 syngeneic mouse cancer model, whereas relativeresistance to anti-PD-1 therapy was preserved in the CT26 and B16F10syngeneic models. Systematic cytokine/chemokine profiling following PD-1blockade in PDOTS from patients with melanoma and other tumorsidentified significant induction of the immunoattractants CCL19 andCXCL13, which was confirmed in vivo and correlated with evidence ofintratumoral immune cell infiltration. Resistance to anti-PD1 treatmentin MDOTS and PDOTS also tracked with coinduction of immune suppressivechemokines. Ex vivo profiling revealed a combination therapy as a noveltherapeutic strategy to enhance sensitivity to PD-1 blockade in thissetting, which effectively predicted tumor response in vivo. Thesefindings demonstrate feasibility of ex vivo profiling of PD-1 blockade,and offer a novel functional approach to facilitate precisionimmunooncology and develop novel therapeutic combinations.

According to one aspect of the invention, a method for evaluating tumorcell spheroids in a three-dimensional microfluidic device is provided.The method involves:

obtaining tumor spheroids from an enzyme treated tumor sample,suspending a first aliquot of the tumor spheroids in biocompatible gel;suspending a second aliquot of the tumor spheroids in biocompatible gel;

placing the first aliquot of the tumor spheroids in biocompatible gel ina first three-dimensional device,

contacting the first aliquot with a first fluorophore dye selective fordead cells, the first fluorophore dye emitting fluorescence at a firstwavelength when bound to a dead cell,

contacting the first aliquot with a second fluorophore dye selective forlive cells, the second fluorophore dye emitting fluorescence at a secondwavelength different from the first wavelength when bound to a livecell,

measuring total fluorescence emitted by each of the first and secondfluorophore dyes in the first aliquot,

culturing the second aliquot in a second three-dimensional device.

contacting the second aliquot with the first fluorophore dye.

contacting the second aliquot with the second fluorophore dye, whereinthe contacting of the second aliquot with the first fluorophore dye andsecond fluorophore dye is carried out at least 24 hours after thecontacting of the first aliquot with the first fluorophore dye andsecond fluorophore dye,

measuring total fluorescence emitted by each of the first and secondfluorophore dyes in the second aliquot,

wherein an increase or decrease in the ratio of live cells to dead cellsin each of the aliquots may be assessed.

In one embodiment, the total fluorescence emitted by each of the firstand second fluorophore dyes is measured using a camera, preferably at aresolution of at least 2×, at least 3×, at least 4× or more, fromdirectly above or below each three-dimensional device, and preferablywherein the three-dimensional devices are placed on a moveable stagepermitting the camera to capture the total fluorescence in each aliquot.

In some embodiments, the dead cell fluorescence and the live cellfluorescence can be added together to yield a live and dead cell total.The amount of dead cells may be expressed as a percentage of the deadcell fluorescence to the total fluorescence. The amount of live cellscan be expressed as a percentage of the live cell fluorescence to thetotal. In some embodiments, the dead cell fluorescence and the live cellfluorescence can be expressed as a ratio, that is, dead cells/live cellsor live cells/dead cells. In this manner, changes to the number of deadcells to live cells can be tracked over time, wherein an initial ratiois established prior to culturing and changes to the ratio aredetermined over time.

In some embodiments, there are multiple aliquots of tumor spheroids, forexample three, or four, or five, or six or more than six, and thecontacting of the third, fourth, fifth, sixth or more than sixth suchaliquots with the first fluorophore dye and second fluorophore dye iscarried out, for example, two days, three days, four days, five days,and more than five days, respectively, after the contacting of the firstaliquot with the first fluorophore dye and second fluorophore dye. Thetiming of testing aliquots may be separated by any time period, such asone day, less than a day, two days or three or more days. In oneembodiment, an aliquot is tested six days after placing the spheroids inthe gel.

In any of the embodiments, the second (or any subsequent) aliquot iscontacted with at least one test compound during the culturing of thesecond aliquot and wherein said culturing of the second aliquot in thepresence of the test compound occurs for at least 24 hours, at least twodays, at least three days, at least four days, at least five days, or atleast 6 days or more.

In any of the foregoing embodiments, the second aliquot can be contactedwith at least two test compounds during the culturing of the secondaliquot and wherein said culturing of the second aliquot in the presenceof the test compounds occurs for at least 24 hours, at least two days,at least three days, at least four days, at least five days, or at least6 days, and preferably wherein at least one of the test compounds is animmune checkpoint inhibitor.

The number of spheroids can be any number of spheroids convenient andavailable to the researcher, but in the system described below, thenumber of spheroids in the first and the second aliquots each containbetween about 15 and 30 spheroids, preferably between about 20 and 25spheroids.

In some embodiments, the first three-dimensional device can be a firstthree-dimensional microfluidic device and the second three-dimensionaldevice can be a second three-dimensional microfluidic device. In someembodiments, the culturing of the first aliquot in the firstthree-dimensional microfluidic device, is for less than 6 hours, lessthan 3 hours, less than 2 hours and even less than 1 hour prior tocontacting the first aliquot with the first and second fluorophore dyes.In particular, it may be desirable to contact the first aliquot with thedyes prior to culturing the spheroids at all, that is essentially uponintroducing the spheroids into the three dimensional device.

In any of the foregoing embodiments, the enzyme can be collagenase.

In any of the foregoing embodiments, the first fluorophore dye can bepropidium iodide, DRAQ7, 7-AAD, eBioscience Fixable Viability DyeeFluor® 455UV, eBioscience Fixable Viability Dye eFluor® 450,eBioscience Fixable Viability Dye eFluor® 506, eBioscience FixableViability Dye eFluor® 520, eBioscience Fixable Viability Dye eFluor®660, eBioscience Fixable Viability Dye eFluor® 780. BioLegend ZombieAqua™, BioLegend Zombie NIR™, BioLegend Zombie Red™, BioLegend ZombieViolet™, BioLegend Zombie UV™, or BioLegend Zombie Yellow™, and/or thesecond fluorophore dye can be acridine orange, nuclear green LCS1(ab138904). DRAQ5 (ab108410), CyTRAK Orange, NUCLEAR-ID Red DNA stain(ENZ-52406), SiR700-DNA, calcein AM, calcein violet AM, calcein blue AM,Vybrant® DyeCycle™ Violet, Vybrant@ DyeCycle™ Green. Vybrant® DyeCycle™Orange, or Vybrant® DyeCycle™ Ruby.

In any of the foregoing embodiments, the tumor spheroids can be obtainedby mincing a primary tumor sample in a medium supplemented with serum;treating the minced primary tumor sample with an enzyme; and harvestingtumor spheroids from the enzyme treated sample. In some embodiments, theminced primary tumor sample is treated with the enzyme in an amountand/or for a time sufficient to yield a partial digestion of the mincedprimary tumor sample, and preferably wherein the treatment is forbetween 10 minutes and 60 minutes, and more preferably between 15minutes and 45 minutes at a temperature of 25° C. to 39° C.

In any of the foregoing embodiments, the biocompatible gel can becollagen, BD Matrigel™ Matrix Basement Membrane, or fibrin hydrogel.

In any of the foregoing embodiments, the tumor sample can be derivedfrom a murine model. In some embodiments, the murine model is asyngeneic model selected from the group consisting of Bladder MBT-2,Breast 4T1, EMT6. Colon, Colon26, CT-26, MC38, Fibrosarcoma WEHI-164,Kidney Renca, Leukemia C1498, L1210, Liver H22, KLN205, LIJ2, LewisLung,Lymphoma A20 S, E.G7-OVA, EL4, Mastocytoma P815. Melanoma B16-BL6,B16-F10, S91, Myeloma MPC-11, Neuroblastoma Neuro-2a, Ovarian: ID8,Pancreatic PanO2, Plasmacytoma J558, and Prostate RM-1.

In any of the foregoing embodiments, the tumor sample can be a humantumor sample.

In any of the foregoing embodiments, the tumor sample can be a patientderived xenograft (PDX).

In any of the foregoing embodiments, the three-dimensional device caninclude one or more fluid channels flanked by one or more gel cageregions, wherein the one or more gel cage regions comprises thebiocompatible gel in which the tumor spheroids are embedded, and whereinthe device recapitulates in vivo tumor microenvironment. In any of theforegoing embodiments, the three-dimensional device can include: asubstrate comprised of an optically transparent material and furthercomprising i) one or more fluid channels; ii) one or more fluid channelinlets; iii) one or more fluid channel outlets; iv) one or more gel cageregions; and v) a plurality of posts; wherein all or a portion of eachgel cage region is flanked by all or a portion of one or more fluidchannels, thereby creating one or more gel cage region-fluid channelinterface regions; each gel cage region comprises at least one row ofposts which forms the gel cage region; and the one or more gel cageregion has a height of less than 500 μm.

In any of the foregoing embodiments, the test compound can be a smallmolecule, a nucleic acid molecule, an RNAi compound, an aptamer, aprotein or a peptide, an antibody or antigen-binding antibody fragment,a ligand or receptor-binding protein, a gene therapy vector, or acombination thereof. In any of the foregoing embodiments, the first testcompound can be a chemotherapeutic compound, an immunomodulatorycompound, or radiation. In any of the foregoing embodiments, the firsttest compound can be an alkylating compound, an antimetabolite, ananthracycline, a proteasome inhibitor, or an mTOR inhibitor. In any ofthe foregoing embodiments, the first test compound can be an immunemodulator.

In some embodiments, the method further comprises isolating RNA from thefirst aliquot and second aliquot of tumor spheroids; and analyzing geneexpression of the first aliquot and second aliquot of tumor spheroidsbased on the isolated RNA, wherein the gene expression is analyzed byperforming RNA sequencing (RNA-seq) on the isolated RNA

In another aspect, provided herein is a method for detecting a change intumor cell spheroids upon exposure to a test compound. The methodcomprises:

culturing a first aliquot of tumor cell spheroids in a firstthree-dimensional device;

culturing a second aliquot of tumor cell spheroids in the presence of afirst test compound in a second three-dimensional device; and

detecting a change in the second aliquot as compared to the firstaliquot, wherein the change is selected from:

a clustering of immune cells around one or more of the tumor cellspheroids of the first or second aliquot;

a decrease in size and/or number of the tumor cell spheroids of thefirst or second aliquot; or

a chemical change.

In some embodiments, the second aliquot is cultured in the presence ofthe first test compound and a second test compound. In some embodiments,the first aliquot is cultured in the presence of the first testcompound.

In some embodiments, the method further comprises:

culturing a third aliquot of tumor cell spheroids in the presence of thefirst test compound and the second test compound in a thirdthree-dimensional device; and

detecting a change in the third aliquot relative to the first and/orsecond aliquot.

In some embodiments, the first aliquot is cultured in the presence ofthe second and/or a third test compound. In some embodiments, the secondaliquot is cultured in the presence of the third and/or a fourth testcompound. In some embodiments, the third aliquot is cultured in thepresence of the third and/or fourth test compound.

In some embodiments, detecting a chemical change comprises detecting apresence of a biological molecule secreted into tumor cell spheroid cellculture supernatant of the first, second, and/or third aliquots.

In some embodiments, the biological molecule is a protein, acarbohydrate, a lipid, a nucleic acid, a metabolite, or a combinationthereof. In some embodiments, the biological molecule is a chemokine ora cytokine. In some embodiments, the biological molecule is known to beassociated with activation of the immune system or otherwise anenhancement of the immune response.

In some embodiments, detecting a chemical change comprises detecting achange in nucleic acid content. In some embodiments, detecting thechange in nucleic acid content comprises detecting a change inextracellular nucleic acids. In some embodiments, detecting the changein nucleic acid content comprises detecting a change in nucleic acidsisolated from tumor cell spheroids from the first, second, and/or thirdaliquots.

In some embodiments, detecting the change in nucleic acid contentcomprises detecting a change in gene expression. In some embodiments,detecting a change in gene expression comprises detecting a changeexpression of genes associated with cytotoxicity. In some embodiments,genes associated with cytotoxicity comprise cytokines and cytokinereceptors.

In some embodiments, detecting the change in nucleic acid contentcomprises analyzing DNA and/or RNA from the first, second, and/or thirdaliquots of tumor cell spheroids. In some embodiments, RNA from thefirst, second, and/or third aliquots of tumor cell spheroids is analyzedby RNA sequencing.

In some embodiments, the first, second, third, and/or fourth testcompound is a small molecule, a nucleic acid molecule, an RNAi compound,an aptamer, a protein or a peptide, an antibody or antigen-bindingantibody fragment, a ligand or receptor-binding protein, a gene therapyvector, or a combination thereof. In some embodiments, the first,second, third, and/or fourth test compound is an immune modulator. Insome embodiments, the immune modulator comprises immune activatingcompounds or inhibitors of an immune checkpoint protein selected fromthe group consisting of CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3 (CD276),B7-H4, 4-1BB (CD137), OX40, ICOS, CD27, CD28, PD-L2, CD80, CD86, B7RP1,HVEM, BTLA, CD137L, OX40L, CD70, CD40, CD40L, GAL9, A2aR, and VISTA. Insome embodiments, the immune checkpoint inhibitor inhibits PD1. In someembodiments, the first test compound is an immune checkpoint inhibitorand the second test compound is a small molecule compound. In someembodiments, the small molecule compound is a TBK-1 inhibitor. In someembodiments, the first, second, third, and/or fourth test compound is achemical from a test compound library. In some embodiments, the firsttest compound is an immune checkpoint inhibitor and the second, third,and/or fourth test compound is a chemical from a test compound library.

In some embodiments, the first, second, and/or third aliquots arecultured in a biocompatible gel. In some embodiments, the first, second,and/or third aliquots are suspended in a biocompatible gel in a fluidchannel of the three-dimensional microfluidic device before culturing.

In some embodiments, the first, second, and/or third aliquots areobtained from an enzyme treated tumor sample.

In another aspect, provided herein is a method for evaluating tumor cellspheroids in a three-dimensional microfluidic device. The methodcomprises:

culturing a first aliquot of tumor spheroids in a firstthree-dimensional device.

culturing the second aliquot of tumor spheroids in a secondthree-dimensional device in the presence of a first test compound;

isolating RNA from the first aliquot and second aliquot of tumorspheroids; and

analyzing gene expression of the first aliquot and second aliquot oftumor spheroids based on the isolated RNA.

In some embodiments, the second aliquot is cultured in the presence of afirst test compound and a second test compound.

In some embodiments, the method further comprises:

culturing the third aliquot of tumor spheroids in a thirdthree-dimensional device in the presence of the first test compound anda second test compound;

isolating RNA from the third aliquot of tumor spheroids; and

analyzing gene expression of the third aliquot of tumor spheroids basedon the isolated RNA.

In some embodiments, the second aliquot is cultured in the presence of athird and/or fourth test compound. In some embodiments, the thirdaliquot is cultured in the presence of a third and/or fourth testcompound.

In some embodiments, the RNA is isolated from the supernatant or fromthe cell culture of the first aliquot and second aliquot of tumorspheroids.

In some embodiments, the gene expression of the first aliquot and secondaliquot of tumor spheroids is analyzed by performing RNA sequencing(RNA-seq) on the isolated RNA.

In some embodiments, the tumor cell spheroids are obtained from anenzyme treated tumor sample.

In some embodiments, the first, second, third, and/or fourth testcompound is a small molecule, a nucleic acid molecule, an RNAi compound,an aptamer, a protein or a peptide, an antibody or antigen-bindingantibody fragment, a ligand or receptor-binding protein, a gene therapyvector, or a combination thereof. In some embodiments, the first testcompound is an immune modulator. In some embodiments, the immunemodulator comprises immune activating compounds or inhibitors of animmune checkpoint protein selected from the group consisting of CTLA-4,PD-1, PD-L1, TIM3, LAG3, B7-H3 (CD276), B7-H4, 4-1BB (CD137), OX40,ICOS, CD27, CD28, PD-L2, CD80, CD86, B7RP1, HVEM, BTLA, CD137L, OX40L,CD70, CD40, CD40L, GAL9, A2aR, and VISTA. In some embodiments, theimmune checkpoint inhibitor inhibits PD1. In some embodiments, the firsttest compound is an immune checkpoint inhibitor and the second testcompound is a small molecule compound. In some embodiments, the smallmolecule compound is a TBK-1 inhibitor. In some embodiments, the first,second, third, and/or fourth test compound is a chemical from a testcompound library. In some embodiments, the first test compound is animmune checkpoint inhibitor and the second, third, and/or fourth testcompound is a chemical from a test compound library.

In some embodiments, the first, second, and/or third aliquots arecultured in a biocompatible gel. In some embodiments, In someembodiments, the first, second, and/or third aliquots are suspended in abiocompatible gel in a fluid channel of the three-dimensionalmicrofluidic device before culturing.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that, in someinstances, various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings.

When describing embodiments in reference to the drawings, directionreferences (“above.” “below,” “top,” “bottom.” “left.” “right,”“horizontal,” “vertical.” etc.) may be used. Such references areintended merely as an aid to the reader viewing the drawings in a normalorientation. These directional references are not intended to describe apreferred or only orientation of an embodied device. A device may beembodied in other orientations.

As is apparent from the detailed description, the examples depicted inthe figures and further described for the purpose of illustrationthroughout the application describe non-limiting embodiments, and insome cases may simplify certain processes or omit features or steps forthe purpose of clearer illustration.

FIG. 1A is a schematic for preparation and analysis of MDOTS/PDOTS (S2fraction) from murine or patient-derived tumor specimens.

FIG. 1B depicts MC38 allograft immune profiling by flow cytometrycomparing bulk tumor (n=5) to S1. S2, S3 (n=6) spheroid fractions(Kruskal-Wallis with Dunn's multiple comparisons test, α=0.05; ns=notsignificant).

FIG. 1C depicts immune profiling of PDOTS (S2: n=40) (upper panel=% livecells, lower panel=% CD45+ cells) with indicated patient/tumorcharacteristics, grouped by tumor type and ranked by % CD8+ T cells.

FIG. 1D depicts immune cell correlation of S2/S3 fractions (CD45, n=14;CD3, n=15; CD4/CD8, n=13; CD4+CD45RO+, n=9; CD8+CD45RO+, n=8;activated=CD38+ and/or CD69+, n=6), R2 significant for all comparisons.

FIG. 1E depicts PD-1. CTLA-4, TIM-3 expression on CD4 and CD8 T cellpopulations in S2/S3 fractions (n=6). R2 significant for allcomparisons.

FIG. 2A depicts phase-contrast imaging (4×) of MC38 MDOTS in 3Dmicrofluidic culture.

FIG. 2B is a cytokine heatmap from cultured MC38 MDOTS expressed aslog-2 fold change relative to Day 1.

FIG. 2C is a cytokine heatmap from cultured B16F10 MDOTS expressed aslog-2 fold change relative to Day 1.

FIG. 2D illustrates MC38 allograft tumor volume following isotypecontrol IgG (n=10) or rat-anti-mouse anti-PD-1 antibody (n=10)treatment.

FIG. 2E illustrates Day 22 MC38 tumor volumes (unpaired 2-sided t-test,p<0.05).

FIG. 2F is a schematic of MDOTS Live/Dead Imaging workflow.

FIG. 2G illustrates Live (AO=green)/dead (PI=red) quantification of MC38MDOTS Day 0 (immediately after loading), Day 3, and Day 6 following IgGcontrol or indicated anti-PD-1 antibody doses; n=4, biologicalreplicates, **p<0.01, ****p<0.0001, Kruskal-Wallis Dunnett's withmultiple comparisons test).

FIG. 2H illustrates Live/dead analysis of MC38 spheroids lacking immunecells±anti-PD1 (n=4, biological replicates).

FIG. 2I illustrates Live/dead analysis of CT26 MDOTS±anti-PD1 (n=3,biological replicates, ****p<0.0001).

FIG. 2J illustrates Live/dead analysis of B16F10 MDOTS t anti-PD1 (n=3,biological replicates).

FIG. 2K depicts deconvolution fluorescence microscopy of MC38 and B16F10MDOTS Day 6±anti-PD1 (representative images shown).

FIG. 3A is a cytokine heatmap that illustrates day 3±anti-PD-1 (n=28)expressed as log 2 fold-change (L2FC) relative to untreated control.

FIG. 3B is a cytokine heatmap that illustrates day 3±anti-CTLA-4 (n=24)expressed as log 2 fold-change (L2FC) relative to untreated control.

FIG. 3C is a cytokine heatmap that illustrates day3±anti-PD-1+anti-CTLA-4 (n=24) expressed as log 2 fold-change (L2FC)relative to untreated control.

FIG. 3D depicts absolute CCL19/CXCL13 levels following single checkpointblockade (2-sided, paired, t-test, α=0.05).

FIG. 3E depicts absolute CCL19/CXCL13 levels following single checkpointblockade (2-sided, paired, t-test, α=0.05).

FIG. 3F depicts absolute CCL19/CXCL13 levels following dual checkpointblockade (2-sided, paired, t-test, α=0.05).

FIG. 3G is a heatmap that illustrates CCL19/CXCL13 mRNA levels frommelanoma biopsy samples on anti-PD1 treatment relative to pre-PD-1(L2FC) by qRT-PCR (n=12).

FIG. 3H is a heatmap that illustrates CCL19/CXCL13 mRNA levels frommelanoma biopsy samples on anti-PD1 treatment relative to pre-PD-1(L2FC) by RNA-seq (n=17 from 10 patients).

FIG. 3I depicts absolute expression (RPKM) for CCL19 and CXCL13 inmelanoma biopsy specimens (pre- and on-treatment) from patients withestablished clinical benefit (CB) or no clinical benefit (NCB) fromcheckpoint blockade.

FIG. 3J depicts absolute expression (RPKM) for CCR7 and CXCR5, therespective receptors for CCL19 and CXCL13, in melanoma biopsy specimens(pre- and on-treatment) from patients with established clinical benefit(CB) or no clinical benefit (NCB) from checkpoint blockade.

FIG. 3K is a heat map that illustrates immune signatures (GSEA) inmelanoma biopsy specimens (pre- and on-treatment) from patients withestablished clinical benefit (CB, n=10 samples from 4 patients) or noclinical benefit (NCB, n=17 samples from 6 patients).

FIG. 3L depicts a Kaplan-Meier survival curve by CCL19/CXCL13 expression(high vs. low).

FIG. 3M depicts a Kaplan-Meier survival curve by four-way sorting usingcutaneous melanoma (SKCM) TCGA data20 (logrank Mantel-Cox test).

FIG. 3N illustrates immune signatures (GSEA) in melanoma biopsyspecimens (pre- and on-treatment) in clusters of patients with varyingexpression of CCL19 and CXCL13 in cutaneous melanoma (SKCM) TCGA.

FIG. 3O is a heatmap that illustrates an unsupervised hierarchicalclustering of Day 3 PDOTS anti-PD1 induced cytokines, expressed as rownormalized (L2FC; n=14), annotated by response to anti-PD-1 therapy andtiming of sample collection.

FIG. 4A depicts a scheme of impact of TBK1/IKKε inhibition on cytokineproduction from tumor cells and T cells.

FIG. 4B depicts the chemical structure of Compound 1 with IC₅₀ towardsTBK1/IKKε, and EC₅₀ in HCT116 cells.

FIG. 4C depicts cytokine heatmaps for CT26 spheroids (lacking immunecells) on Day 1, 3, and 6±Compound 1 (n=3, biological replicates)expressed as log 2 fold-change (L2FC) relative to vehicle control.

FIG. 4D is a dose-response curve for Compound 1 on IL-2 in human CD4(n=3) and CD8 (n=5) T cells.

FIG. 4E is a dose-response curve for Compound 1 on IFNγ in human CD4(n=3) and CD8 (n=5) T cells.

FIG. 4F depicts cytokine heatmaps for CT26 MDOTS treated with IgG+Cmpd1(1 μM), αPD-1 (10 μg/mL), or αPD-1+Cmpd1 (1 μM) from the mean of n=3biological replicates, plotted as L2FC relative to isotype control IgGwith vehicle control. 2-sided Welch's 2-sample t-test with unequalvariance (α=0.05).

FIG. 4G is a chart that depicts live (AO=green)/dead (PI=red)quantification of CT26 MDOTS after 6 days treated with IgG-DMSO, Cmpd1(1 μM), αPD-1, and αPD-1+Cmpd1 (*p<0.05, Kruskal-Wallis ANOVA withmultiple comparisons; n=3).

FIG. 4H depicts imaging results corresponding to live (AO=green)/dead(PI=red) quantification of CT26 MDOTS after 6 days treated withIgG-DMSO, Cmpd1 (1 μM), αPD-1, and αPD-1+Cmpd1 (*p<0.05, Kruskal-WallisANOVA with multiple comparisons; n=3).

FIG. 4I depicts CT26 allograft tumor volume over time followingIgG+vehicle. IgG+Cmpd1, αPD-L1+vehicle, and αPD-L1+Cmpd1 (n=10 pergroup, **p<0.01, 1-way ANOVA with Tukey's multiple comparison's test fortumor volume, log-rank Mantel-Cox test for Kaplan-Meier analysis forentire group and pairwise comparisons).

FIG. 4J depicts CT26 allograft percent change in tumor volume followingIgG+vehicle. IgG+Cmpd1, αPD-L1+vehicle, and αPD-L1+Cmpd1 (n=10 pergroup, **p<0.01, 1-way ANOVA with Tukey's multiple comparison's test fortumor volume, log-rank Mantel-Cox test for Kaplan-Meier analysis forentire group and pairwise comparisons).

FIG. 4K depicts CT26 allograft percent survival following IgG+vehicle,IgG+Cmpd1, αPD-L1+vehicle, and αPD-L1+Cmpd1 (n=10 per group, **p<0.01,1-way ANOVA with Tukey's multiple comparison's test for tumor volume,log-rank Mantel-Cox test for Kaplan-Meier analysis for entire group andpairwise comparisons).

FIG. 5A depicts common gating for viable CD45+ cells in immune cellprofiling by multi-color flow cytometry experiments.

FIG. 5B depicts gating hierarchy for T cell lineage and phenotypicmarker in immune cell profiling by multi-color flow cytometryexperiments.

FIG. 5C depicts myeloid cell gating hierarchy in immune cell profilingby multi-color flow cytometry experiments.

FIG. 5D depicts gating for NK lineage and Tregs in immune cell profilingby multi-color flow cytometry experiments.

FIG. 6A depicts immune cell sub-populations in MC38 tumors and/orspheroids from immune cell profiling of MDOTS. Immune cellsub-populations from MC38 (a) bulk tumor (n=5) to S1, S2, S3 (n=6) wereevaluated by flow cytometry, as in FIG. 1b (Kruskal-Wallis test withDunn's multiple comparisons test, α=0.05: *p<0.05; ns=not significant).

FIG. 6B depicts a Pearson correlation matrix using composite of 21 cellsurface markers in MC38 tumor, S1, S2, and S3 spheroids.

FIG. 6C depicts immune cell sub-populations in B16F10 tumors and/orspheroids from immune cell profiling of MDOTS. Immune cellsub-populations from B16F10 (a) bulk tumor (n=5) to S1 (n=4), S2 (n=5),and S3 (n=4) evaluated by flow cytometry (Kruskal-Wallis test withDunn's multiple comparisons test. α=0.05; *p<0.05; ns=not significant).

FIG. 6D depicts CD45+ cells (% live cells) in MC38 (n=6), B16F10 (n=5),and CT26 (n=5) MDOTS (S2). Kruskal-Wallis test with Dunn's multiplecomparisons test, α=0.05; *p<0.05.

FIG. 6E depicts immune sub-populations (% CD45+ cells) in MC38 (n=6),B16F10 (n=5), and CT26 (n=5) MDOTS (S2). 2-way ANOVA with Tukey'smultiple comparisons test, α=0.05; *p<0.05, ***p<0.001, ****p<0.0001).

FIG. 6F depicts surface expression of T cell exhaustion markers (PD-1,CTLA-4, TIM-3) on CD4 and CD8 populations in PDOTS (n=40) from immunecell profiling of PDOTS.

FIG. 6G depicts surface expression of PD-L1 and PD-L2 on myeloidsub-populations, including granulocytic myeloid-derived suppressor cells(gMDSCs), monocytes, tumor-associated macrophages (TAMs), monocyticmyeloid-derived suppressor cells (mMDSCs), and plasmacytoid dendriticcells (pDCs).

FIG. 6H depicts immune cell sub-populations of PDOTS fractions (S1, S2,S3) evaluated by flow cytometry, for CD45+(n=8), CD3+(n=8), CD4+(n=7),CD8+(n=7), CD14+(n=7), and CD15+(n=6). Kruskal-Wallis test with Dunn'smultiple comparisons test, α=0.05; ns=not significant.

FIG. 6I depicts antigen-experienced sub-population (CD45RO+), effectormemory (CD45RO+CCR7−) subtype, and surface expression of T cellexhaustion markers (PD-1, CTLA-4, TIM-3) on CD4 populations in PDOTSfractions (n>3; Kruskal-Wallis test with Dunn's multiple comparisonstest, α=0.05; ns=not significant).

FIG. 6J depicts antigen-experienced sub-population (CD45RO+), effectormemory (CD45RO+CCR7−) subtype, and surface expression of T cellexhaustion markers (PD-1, CTLA-4, TIM-3) on CD8 populations in PDOTSfractions (n≥3; Kruskal-Wallis test with Dunn's multiple comparisonstest, α=0.05; ns=not significant).

FIG. 7A depicts immunofluorescence staining of MC38 MDOTS.

FIG. 7B depicts immunofluorescence staining of NSCLC PDOTS.

FIG. 7C depicts immunofluorescence staining of NSCLC PDOTS.

FIG. 7D depicts immunofluorescence staining for MC38 MDOTS stained forCD45+ immune cells (purple) and CD8+ T cells (yellow) with calcein(green) staining live cells and Hoechst (blue) staining all cell nucleiat Day 7 after treatment with IgG (10 μg/mL) or anti-PD-1 (10 μg/mL)(scale bar=20 μm).

FIG. 7E is a chart that depicts live/dead analysis of MC38 MDOTS tanti-PD-1 performed by independent lab (n=3, biological replicates).

FIG. 7F is a chart that depicts live/dead analysis of CT26 MDOTSperformed on Day 6 following treatment with isotype IgG control (10μg/mL) or anti-PD-1 (10 μg/mL) t anti-CD8 (10 μg/mL) (n=6, biologicalreplicates; 2-way ANOVA with Tukey's multiple comparisons test;****p<0.0001, ns=not significant).

FIG. 7G depicts a comparison of intratumoral and inter-tumoralheterogeneity evaluating CD8+ T cell counts (IF, performed on Day 4) andlive/dead analysis (AO/PI staining) of CT26 MDOTS (Day 5).

FIG. 8A depicts absolute cytokine levels (pg/mL) obtained by bead-basedcytokine profiling of PDOTS under control conditions or in response toαPD-1 grouped by Th1 and IFN-γ effector cytokines. (n=28; 2-sided,paired, t-test, α=0.05).

FIG. 8B depicts absolute cytokine levels (pg/mL) obtained by bead-basedcytokine profiling of PDOTS under control conditions or in response toαPD-1 grouped by granulocyte chemoattractants, (n=28; 2-sided, paired,t-test, α=0.05).

FIG. 8C depicts absolute cytokine levels (pg/mL) obtained by bead-basedcytokine profiling of PDOTS under control conditions or in response toαPD-1 grouped by IPRES immune suppressive cytokines, (n=28; 2-sided,paired, t-test, α=0.05).

FIG. 8D illustrates a comparison of PDOTS cytokine profiles after 3 daysin standard growth conditions with and without isotype control IgGantibody (50 μg/mL) (n=2), performed using PDOTS derived from sampleMGH-16 (melanoma).

FIG. 8E depicts CCL19 and CXCL13 levels (pg/mL) relative to the numberof spheroids per device in control and treatment (αPD-1) conditions(R2=Pearson correlation coefficient).

FIG. 8F depicts results of biological replicates for MGH-16 PDOTS inresponse to PD-1 blockade (n=3, L2FC relative to untreated control onDay 3).

FIG. 8G depicts CCL19 and CXCL13 levels (pg/mL) from biologicalreplicates for MGH-16 PDOTS in response to PD-1 blockade (n=3. L2FCrelative to untreated control on Day 3).

FIG. 8H depicts the correlation between CCL9 and CXCL13 upregulation(log 2 fold-change relative to untreated control) in response to αPD-1,αCTLA-4, or αPD-1+αCTLA-4 (R2=Pearson correlation coefficient).

FIG. 8I illustrates the effect of culture in the microfluidic device oncytokine profile following αPD-1 treatment. Equal volumes of PDOTS frompatient MGH-16 (in collagen hydrogels) were loaded for microfluidicdevice (or into a single well of a 96-well plate in equal volumes ofculture media. Media was collected on Day 3 (control and αPD-1) forcytokine profiling. Induction of cytokines represented as fold-changerelative to the untreated control.

FIG. 9A is a heatmap demonstrating log(2) fold-change (relative tountreated control, ranked highest to lowest) in cytokine profiles usingconditioned media obtained at indicated time points of ex vivomicrofluidic culture with αPD-1, αCTLA-4, or αPD-1+αCTLA-4 in DFCI-25(melanoma PDOTS). Arrows denote effector cytokines (e.g. IFN-γ, IL-2)associated with immune-mediated cytolysis.

FIG. 9B is a heatmap demonstrating log(2) fold-change (relative tountreated control, ranked highest to lowest) in cytokine profiles usingconditioned media obtained at indicated time points of ex vivomicrofluidic culture with αPD-1, αCTLA-4, or αPD-1+αCTLA-4 in MGH-12(melanoma PDOTS). Arrows denote effector cytokines (e.g. IFN-γ, IL-2)associated with immune-mediated cytolysis.

FIG. 9C is a heatmap demonstrating log(2) fold-change (relative tountreated control, ranked highest to lowest) in cytokine profiles usingconditioned media obtained at indicated time points of ex vivomicrofluidic culture with αPD-1. αCTLA-4, or αPD-1+αCTLA-4 in DFCI-16(thyroid carcinoma PDOTS). Arrows denote effector cytokines (e.g. IFN-γ,IL-2) associated with immune-mediated cytolysis.

FIG. 9D is a heatmap demonstrating log(2) fold-change (relative tountreated control, ranked highest to lowest) in cytokine profiles usingconditioned media obtained at indicated time points of ex vivomicrofluidic culture with αPD-1, αCTLA-4, or αPD-1+αCTLA-4 in DFCI-19(pancreatic adenocarcinoma PDOTS). Arrows denote effector cytokines(e.g. IFN-γ, IL-2) associated with immune-mediated cytolysis.

FIG. 10A depicts RNA-seq showing select cytokines, cytokine receptors,cytotoxic T cell (CTL) associated genes, and IPRES transcripts (L2FCrelative to pre-treatment control in 10 sets of patient samples).

FIG. 10B depicts RNA-seq absolute expression (RPKM) for cytotoxic T celleffector associated genes (IFNG, IFNGR1, granzyme A, granzyme B) frompatients with established clinical benefit (CB, n=10 samples from 4patients) or no clinical benefit (NCB, n=17 samples from 6 patients)from checkpoint blockade.

FIG. 10C depicts RNA-seq absolute expression (RPKM) for select IPRESgenes in melanoma biopsy specimens (pre- and on-treatment) from patientswith established clinical benefit (CB, n=10 samples from 4 patients) orno clinical benefit (NCB, n=17 samples from 6 patients) from checkpointblockade.

FIG. 10D depicts pre-treatment expression of CCL19 and CXCL13 inresponders (n=15) and non-responders (n=13) to PD-1 blockade. Expressiondata is represented as transcripts per million (TPM) with error barsindicating standard deviation (ns=not significant; unpaired, 2-sided,Mann-Whitney test, α=0.05).

FIG. 10E depicts pre-treatment expression of CCL19 and CXCL13 inpatients who experienced clinical benefit (CB, n=14), no clinicalbenefit (NCB, n=22), and no clinical benefit, but long-term survival(NCB+LTS, n=5) from ipilimumab (αCTLA-4). Expression data is representedas transcripts per million (TPM) with error bars indicating standarddeviation (ns=not significant; Kruskal-Wallis with Dunn's multiplecomparisons test, α=0.05).

FIG. 10F is a heatmap demonstrating log(2) fold change(on-αPD-1/pre-αPD-1) in expression of indicated cytokines.

FIG. 10G is a heatmap demonstrating log(2) fold change of plasmacytokines using matched plasma samples (on-treatment/pre-treatment) forresponders (R; n=7) and non-responders (NR; n=4) to anti-PD-1 therapy.

FIG. 10H depicts four-way Kaplan-Meier survival curves by CCL19/CXCL13expression (high-high, high-low, low-high, and low-low) using urothelialbladder carcinoma (BLCA), head & neck squamous cell carcinoma (HNSC),and breast carcinoma (BRCA) TCGA data (pairwise analysis using log-rankMantel-Cox test, *p<0.05).

FIG. 11A depicts H&E imaging (10× objective) of tumor-associatedtertiary lymphoid structure (TA-TLS), higher magnification (40×objective), and quantitation of TA-TLS identified in 52 H&E slides.

FIG. 11B depicts PDOTS divided into CCL19/CXCL13-high (n=14) andCCL19/CXCL13-low (n=14) by median L2FC (Mann-Whitney test,****p<0.0001).

FIG. 11C depicts median immune cell composition (CD45+, CD4+, CD8+,CD14+, CD15+) between PDOTS with distinct ex vivo expression ofCCL19/CXCL13 (high vs. low).

FIG. 11D depicts median immune cell composition (CD4+PD1+ and CD8+PD-1+)in CCL19/CXCL13 high vs. low PDOTS.

FIG. 11E depicts relative expression of CCL19 and CXCL13 by qRT-PCR incancer-associated fibroblasts (CD45−CD90+), cancer-associatedendothelial cells (CD31+CD144+), CD45− (cancer cells, etc.) and CD45+immune cells following 4-way cell sorting (n=2, technical replicatesfrom MGH-16).

FIG. 11F depicts immunohistochemical staining of CCL19, CXCL13, αSMA(cancer-associated fibroblasts), and CD31 (endothelial cells) inmelanoma specimen (pt 422, on-treatment; 40× magnification, scale bar400 μm).

FIG. 12A depicts mean cytokine changes from PDOTS following ex vivo PD-1blockade in patients with clinical benefit (n=5: PR/SD) and withoutclinical benefit (n=9; MR/PD) from PD-1 blockade (*p<0.05).

FIG. 12B depicts four-way Kaplan-Meier survival curves by CX3CL1/CCL20expression (high-high, high-low, low-high, and low-low) using melanomaTCGA data (pairwise analysis using log-rank Mantel-Cox test, α=0.05,ns=not significant).

FIG. 12C depicts composite IPRES6 score (sum of L2FC for CCL2, CCL7,CCL8, CCL13, IL-10) in response to ex vivo PD-1 blockade in PDOTSsamples from patients with clinical benefit (PR/SD) and without clinicalbenefit (MR/PD) from PD-1 blockade.

FIG. 12D depicts heatmaps of Day 3 PDOTS anti-PD1 induced cytokines forsamples DFCI-13, -16, -18, and -21 (serial sampling from same patientwith metastatic papillary thyroid cancer), expressed as L2FC relative tountreated control.

FIG. 12E is a chart that depicts serial immune profiling of samplesDFCI-13, -16, -18, -21, and -22 from the same patient undergoingindicated treatments.

FIG. 12F is a heatmap of relative anti-PD1 induced cytokine changes fromMC38 and B16F10 MDOTS (L2FC relative to isotype control; n=3, biologicalreplicates).

FIG. 12G is a chart that depicts absolute cytokine levels in CT26 MDOTSover time (n=3, biological replicates).

FIG. 12H depicts Live/Dead analysis at Day 6 in CT26 MDOTS treated withanti-CCL2±anti-PD-1 at Days 1, 3, and 6 (L2FC relative to isotypecontrol; n=3, biological replicates).

FIG. 12I is a heatmap of relative cytokine changes in CT26 MDOTS treatedwith anti-CCL2±anti-PD-1 at Days 1, 3, and 6 (L2FC relative to isotypecontrol; n=3, biological replicates).

FIG. 13A is a scheme that depicts chemical synthesis of Compound 1.

FIG. 13B is a chart that depicts IC₅₀ values for indicated enzymestreated with Compound 1.

FIG. 13C depicts an IC₅₀ curve for TBK1.

FIG. 13D depicts an IC₅₀ curve for IKKε (IKBKE).

FIG. 13E is a chart that depicts effect of Compound 1 on viability ofCT26 spheroids lacking immune cells (n=3, biological replicates).

FIG. 13F illustrates fold-change in IL-2 levels produced by Jurkat cellswith increasing doses of Compound 1.

FIG. 13G depicts change in tumor volume following re-implantation ofCT26 and EMT-6 cells into the flanks of mice previously treated withanti-PD-L1+ Compound 1.

FIG. 14A is a cytokine heatmap for MC38 MDOTS treated with IgG+Cmpd1 (1μM), αPD-1 (10 μg/mL), or αPD-1+Cmpd1 (1 μM) from the mean of n=3biological replicates, plotted as L2FC relative to isotype control IgG(10 μg/mL) with vehicle control (DMSO 0.1%). 2-sided Welch's 2-samplet-test with unequal variance (α=0.05).

FIG. 14B is a cytokine heatmap for B16F10 MDOTS treated with IgG+Cmpd1(1 μM), αPD-1 (10 μg/mL), or αPD-1+Cmpd1 (1 μM) from the mean of n=3biological replicates, plotted as L2FC relative to isotype control IgG(10 μg/mL) with vehicle control (DMSO 0.1%). 2-sided Welch's 2-samplet-test with unequal variance (α=0.05).

FIG. 14C is a chart that depicts live (AO=green)/dead (PI=red)quantification of MC38 MDOTS after 5 days treated with IgG-DMSO, Cmpd1(1 μM), αPD-1, and αPD-1+Cmpd1 (*p<0.05, Kruskal-Wallis ANOVA withmultiple comparisons; n=3).

FIG. 14D depicts MC38 allograft tumor volume waterfall plot followingIgG+vehicle, IgG+Cmpd1, αPD-L1+vehicle, and αPD-L1+Cmpd1 (n=10 pergroup, *p<0.05, **p<0.01, 1-way ANOVA with Tukey's multiple comparison'stest for tumor volume, log-rank Mantel-Cox test for Kaplan-Meieranalysis for entire group and pairwise comparisons).

FIG. 14E depicts percent survival following IgG+vehicle, IgG+Cmpd1,αPD-L1+vehicle, and αPD-L1+Cmpd1 (n=10 per group, *p<0.05, **p<0.01,1-way ANOVA with Tukey's multiple comparison's test for tumor volume,log-rank Mantel-Cox test for Kaplan-Meier analysis for entire group andpairwise comparisons).

FIG. 14F is a chart that depicts live (AO=green)/dead (PI=red)quantification of B16F10 MDOTS after 6 days treated with IgG-DMSO, Cmpd1(1 μM), αPD-1, and αPD-1+Cmpd1 (α=0.05, ns=not significant.Kruskal-Wallis ANOVA with multiple comparisons; n=3).

FIG. 14G depicts quantification of small lung metastases in B16F10 tailvein injection model following IgG+vehicle, IgG+Cmpd1, αPD-L1+vehicle,and αPD-L1+Cmpd1 (n=10 per group, 1-way ANOVA with Tukey's multiplecomparison's test, ns=not significant).

FIG. 14H depicts quantification of medium lung metastases in B16F10 tailvein injection model following IgG+vehicle, IgG+Cmpd1, αPD-L1+vehicle,and αPD-L1+Cmpd1 (n=10 per group, 1-way ANOVA with Tukey's multiplecomparison's test, ns=not significant).

FIG. 14I depicts MB49 allograft tumor volume waterfall plot followingIgG+vehicle, IgG+Cmpd1, αPD-L1+vehicle, and αPD-L1+Cmpd1 (n=10 pergroup, *p<0.05, 1-way ANOVA with Tukey's multiple comparison's test fortumor volume, log-rank Mantel-Cox test for Kaplan-Meier analysis forentire group and pairwise comparisons).

FIG. 14J depicts MB49 allograft tumor volume waterfall plot as a barchart following IgG+vehicle, IgG+Cmpd1, αPD-L1+vehicle, and αPD-L1+Cmpd1(n=10 per group, *p<0.05, 1-way ANOVA with Tukey's multiple comparison'stest for tumor volume, log-rank Mantel-Cox test for Kaplan-Meieranalysis for entire group and pairwise comparisons).

FIG. 14K depicts percent survival following IgG+vehicle, IgG+Cmpd1,αPD-L1+vehicle, and αPD-L1+Cmpd1 (n=10 per group, *p<0.05, 1-way ANOVAwith Tukey's multiple comparison's test for tumor volume, log-rankMantel-Cox test for Kaplan-Meier analysis for entire group and pairwisecomparisons).

FIG. 15 depicts a table summary of Pilot RNA Extraction, QC, andQuantitation (left), and a representation bioanalyzer trac showing fullyintact total RNA from MDOTS (right).

FIG. 16A is a sample-sample correlation of RNA-seq data collected from avariety of MC38 MDOTS treatment types (αPD-1, αPD-1+CD8, IFNγ, IgG,PD-1) at two distinct timepoints (Day 3. Day 6).

FIG. 16B provides two depictions of a principal components (PC) analysisof RNA-seq data collected from a variety of MC38 MDOTS treatment types(αPD-1, αPD-1+CD8, IFNγ, IgG, PD-1) at two distinct timepoints (Day 3.Day 6).

FIG. 16C depicts RNA-seq data collected from MC38 MDOTS sample treatedwith IFNγ on Day 6. Of note, several IFNγ genes are induced, includingCXCL10, CXCL11, PD-L1, IDO1, and IDO2.

FIG. 16D depicts RNA-seq data collected from MC38 MDOTS sample treatedwith αPD-1 on Day 6. Of note, several novel targets and biomarkers areinduced, including SOCS3, Pde4b, Pde4d, and Adcy5.

DETAILED DESCRIPTION

Among other aspects, the disclosure provides methods for evaluatingtumor cell spheroids in a three-dimensional microfluidic device bymonitoring a ratio of live cells to dead cells. In some aspects, themethods include steps of exposing separate aliquots of a tumor spheroidsample to different conditions, such as the inclusion or exclusion of atest compound. As such, in some embodiments, the effects of a testcompound can be evaluated by monitoring its effects on the ratio of livecells to dead cells. It has been discovered, surprisingly, that thetumor microenvironment of a cultured tumor spheroid can be evaluatedoptically and reproducibly, to establish the effects of drugs on thetumor cells within the spheroids, while distinguishing the effects ontumor cells resulting from the act of culturing the tumor cells. Alsoprovided are other methods for evaluating tumor sphere spheroids,including monitoring a chemical change, e.g., such as a change innucleic acid expression, when separate aliquots of a tumor spheroidsample are exposed to different conditions, such as the inclusion orexclusion of a test compound.

As used herein, the term “tumor” refers to a neoplasm. i.e., an abnormalgrowth of cells or tissue and is understood to include benign, i.e.,non-cancerous growths, and malignant; i.e., cancerous growths includingprimary or metastatic cancerous growths.

Examples of neoplasms include, but are not limited to, mesothelioma,lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer),skin cancer (e.g., melanoma), stomach cancer, liver cancer, colorectalcancer, breast cancer, pancreatic cancer, prostate cancer, blood cancer,bone cancer, bone marrow cancer, and other cancers.

The term “tumor spheroid,” or “tumor cell spheroid” as used herein,refers to an aggregation of tumor cells constituting a small mass, orlump of tumor cells. In some embodiments, tumor spheroids are less thanabout 3 cm, less than about 2 cm, less than about 1 cm, less than about5 mm, less than about 2.5 mm, less than about 1 mm, less than about 100μm, less than about 50 μm, less than about 25 μm, less than about 10 μm,or less than about 5 μm in diameter. In some embodiments, the tumorspheroids have a diameter of 10 μm to 500 μm. In some embodiments, thetumor spheroids have a diameter of 40 μm to 100 μm. In some embodiments,the tumor spheroids have a diameter of 40 μm to 70 μm.

The term “primary tumor sample” as used herein refers to a samplecomprising tumor material obtained from a subject having cancer. Theterm encompasses tumor tissue samples, for example, tissue obtained bysurgical resection and tissue obtained by biopsy, such as for example, acore biopsy or a fine needle biopsy. The term also encompasses patientderived xenograft (PDX). Patient derived xenografts are created whencancerous tissue from a patient's primary tumor is implanted directlyinto an immunodeficient mouse (see, for example. Morton C L, Houghton PJ (2007). “Establishment of human tumor xenografts in immunodeficientmice”. Nature Protocols 2 (2): 247-50; Siolas D. Hannon G J (September2013). “Patient-derived tumor xenografts: transforming clinical samplesinto mouse models”. Cancer Research 73 (17): 5315-9). PDX mirrorspatients' histopathological and genetic profiles. It has improvedpredictive power as preclinical cancer models, and enables the trueindividualized therapy and discovery of predictive biomarkers.

In some embodiments, the subject is a human. In some embodiments, thesubject is a non-human mammal or a non-human vertebrate. In someembodiments, the subject is laboratory animal, a mouse, a rat, a rodent,a farm animal, a pig, a cattle, a horse, a goat, a sheep, a companionanimal, a dog a cat, or a guinea pig.

In some embodiments, a tumor sample as used herein is derived from asubject known to have resistance or sensitivity to a particulartherapeutic treatment. For example, in some embodiments, methodsprovided herein can identify novel combination therapies and/or novelsingle therapies that overcome resistance to a specified treatment.e.g., PD-1 inhibition in the treatment of cancer. In some embodiments,where PD-1 combination therapies are to be evaluated, tumor samples maybe derived from one or more subjects having the same or varied levels ofsensitivity to PD-1 inhibition therapy. In some embodiments, the tumorsample is derived from a B16F10 murine model. B16F10 is a murine modelthat is minimally sensitive to PD-1 blockade. In some embodiments, thetumor sample is derived from an MC38 murine model. MC38 is a murinemodel that is highly sensitive to PD-1 blockade relative to B16F10. Insome embodiments, the tumor sample is derived from a CT26 murine model.CT26 is a murine model that is of an intermediate sensitivity relativeto B16F10 and MC38 models. In some embodiments, methods provided hereincomprises steps of evaluating a combination of two or more of tumorsamples derived from B16F10, MC38, or CT26 models.

Tumor cell spheroids can be prepared by any method known in the art.Exemplary methods for preparing tumor cell spheroids are described in WO2016/112172, the disclosure of which is incorporated herein byreference.

In some embodiments, the primary tumor sample is collected in aserum-supplemented medium, for example but not limited to, RPMI mediumsupplemented with 10% fetal bovine serum. The sample is then minced,i.e., cut or chopped into tiny pieces. In some embodiments, the sampleis minced on ice. In some embodiments, the minced primary tumor samplecomprises tumor pieces in the size of about 3 mm, 2.5 mm, 2.0 mm, 1.5mm, 1.0 mm, 0.5, or 0.25 mm.

In some embodiments, the primary tumor sample is not frozen and thawed.

In some embodiments, minced primary tumor sample is frozen in a mediumsupplemented with serum and thawed prior to treating with thecomposition comprising the enzyme. In some embodiments, the mincedprimary tumor sample is frozen for at least 6 hours 12 hours, 24 hours,2 days, 1 week or one month. In some embodiments, the minced primarytumor sample is frozen at −80° C. In some embodiments, the mincedprimary tumor sample is frozen in liquid nitrogen. In some embodiments,the minced primary tumor sample is frozen in a medium supplemented withserum. In some embodiments, the minced primary tumor sample is frozen ina mixture containing serum and solvent such as Dimethyl sulfoxide(DMSO). In some embodiments, the minced primary tumor sample is frozenin a mixture containing fetal bovine serum and Dimethyl sulfoxide(DMSO).

In some embodiments, the frozen minced primary tumor sample is thawed,i.e., defrosted, before treating the sample with a compositioncomprising an enzyme. In some embodiments, the minced primary tumorsample is thawed in a water bath kept at about 37° C. (e.g., 35° C., 36°C., 37° C., 38° C., or 39° C.). In some embodiments, the minced primarytumor sample is thawed at room temperature.

The minced primary tumor sample is treated with an enzyme mix to digestthe tumor samples. In some embodiments, the composition comprising anenzyme includes collagenase. In some embodiments, the compositioncomprising an enzyme includes a serum-supplemented culture medium,insulin, one or more corticosteroids, one or more antibiotics,collagenase and optionally one or more growth factors.Serum-supplemented culture media, corticosteroids, antibiotics, andgrowth factors are well-known in the art. In some embodiments, thecomposition comprising an enzyme comprises DMEM or RPMI, fetal bovineserum, insulin, epidermal growth factor, hydrocortisone, Penicillinand/or Streptomycin, and collagenase. In some embodiments, thecomposition comprising an enzyme comprises further comprises a bufferingagent such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES). “Treating the minced primary tumor sample with a compositioncomprising an enzyme” comprises incubating the minced tumor samples withthe enzyme composition for at least 1 hour.

In some embodiments, the minced tumor samples are incubated with theenzyme mix for at least 2 hours, at least 4 hours, at least 6 hours, atleast 8 hours, at least 10 hours, at least 12 hours, at least 15 hoursor at least 24 hours. In some embodiments, the minced primary tumorsample is incubated with the enzyme mix at 25° C., 26° C., 27° C., 28°C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37°C. 38° C., or 39° C. In some embodiments, the minced primary tumorsample is incubated with the enzyme mix at 37° C.

In some embodiments, the minced primary tumor sample is treated with thecomposition comprising the enzyme in an amount or for a time sufficientyield a partial digestion of the minced primary tumor sample. In someembodiments, the minced primary tumor sample is treated with thecomposition comprising the enzyme for 30 minutes to 15 hours at atemperature of 25° C. to 39° C.

Collecting tumor spheroids from the enzyme mix treated sample comprisescentrifuging and washing the sample at least twice followed by isolatingthe digested tumor spheroids of the desired size. In some embodiments,the enzyme mix treated sample is centrifuged and washed using phosphatebuffered saline (PBS) at least twice. Tumor spheroids of the desiredsize are collected using sieves. In some embodiments, the tumorspheroids having a diameter of 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400μm, 450 μm, and 500 μm are collected from the enzyme mix treated samplewith the use of a sieve. In some embodiments, the tumor spheroids havinga diameter of 40 μm to 100 μm are collected from the enzyme mix treatedsample with the use of a sieve. In some embodiments, the tumor spheroidshaving a diameter of 40 μm, 50 μm, 60 μm and 70 μm are collected fromthe enzyme mix treated sample with the use of a sieve.

The tumor spheroids having a desired diameter are collected by sievingthe enzyme mix treated sample through cell strainers. In someembodiments, the tumor spheroids having a diameter of 10 μm to 500 μmare collected by sieving the enzyme mix treated sample via 500 μm and 10μm cell strainers to yield tumor spheroids having a diameter of 10 μm to500 μm. In some embodiments, the tumor spheroids having a diameter of 40μm to 100 μm are collected by sieving the enzyme mix treated sample via100 μm and 40 μm cell strainers to yield tumor spheroids having adiameter of 10 μm to 500 μm. The tumor spheroids of the desired diameterare collected and suspended in a biocompatible gel. Examples ofbiocompatible gel include collagen, BD Matrigel™ Matrix BasementMembrane, or fibrin hydrogel (e.g., fibrin hydrogel generated fromthrombin treatment of fibrinogen).

In some embodiments, the collected tumor spheroids are not frozen andthen thawed before suspending in the biocompatible gel. In someembodiments, the collected tumor spheroids are introduced into thethree-dimensional cell culture device within less than 2 hours, lessthan 1 hour, less than 30 minutes, less than 20 minutes, less than 10minutes, or less after collection.

In some embodiments, the collected tumor spheroids are frozen in afreezing medium and then thawed before suspending in the biocompatiblegel. In some embodiments, the collected tumor spheroids are frozen forat least 6 hours 12 hours, 24 hours, 2 days, 1 week or one month. Insome embodiments, the collected tumor spheroids are frozen at −80° C. Insome embodiments, the collected tumor spheroids are frozen in liquidnitrogen. In some embodiments, the collected tumor spheroids are frozenat −80° C. overnight, and then transferred to liquid nitrogen forstorage. In some embodiments, the collected tumor spheroids are frozenin a medium supplemented with serum. In some embodiments, the collectedtumor spheroids are frozen in a mixture containing culture medium suchas DMEM or RPMI, fetal bovine serum and solvent such as Dimethylsulfoxide (DMSO). The frozen spheroids are thawed, for example overnightat 4° C., and then suspended in the biocompatible gel.

The tumor spheroids are cultured, i.e., grown, in a three dimensional(3D) microfluidic device. In some embodiments, the tumor spheroids arecultured with endothelial cells, such as human umbilical veinendothelial cells (HUVECs). In some embodiments, the tumor spheroids arecultured without endothelial cells. In some embodiments, the tumorspheroids are cultured with or without endothelial cells for at least 1day, at least 2 days, at least 4 days, at least 6 days, at least 1 week,or at least 2 weeks.

3D microfluidic devices are known in the art and include, for example,but not limited to, the devices described in US 2013/0143230, EP2741083,US 2014/0057311, U.S. Pat. No. 8,748,180, and WO 2016/112172, thedisclosures of which are incorporated by reference herein.

In some embodiments, a 3D microfluidic device refers to a device thatcomprises one or more fluid channels flanked by one or more gel cageregions, wherein the one or more gel cage regions comprises thebiocompatible gel in which the tumor spheroids are embedded, and whereinthe device recapitulates, i.e., mimics, the in vivo tumormicroenvironment. In order to facilitate visualization, the microfluidicdevice is typically comprised of a substrate that is transparent tolight, referred to herein as “an optically transparent material”. Aswill be appreciated by those of skill in the art, suitable opticallytransparent materials include polymers, plastic, and glass. Examples ofsuitable polymers are polydimethylsiloxane (PDMS), poly(methylmethacrylate) (PMMA), polystyrene (PS), SU-8, and cyclic olefincopolymer (COC). In some embodiments, all or a portion of the device ismade of a biocompatible material, e.g., the material is compatible with,or not toxic or injurious to, living material (e.g., cells, tissue).

The fluid channel can be used to contain a (one or more) fluid (e.g.,cell culture media), cells such as endothelial cells, cellular material,tissue, fluorophore dyes, and/or compounds (e.g., drugs) to be assessed,while the gel cage regions may be used to contain a gel (e.g.,biologically relevant gel, such as collagen, Matrigel™, or fibrinhydrogel (e.g., fibrin hydrogel generated from thrombin treatment offibrinogen)). In some embodiments, the 3D microfluidic device comprisesthe device described in US 2014/0057311, the disclosure of which isincorporated by reference herein. In particular, paragraphs [0056] to[0107] which describe the regions, channels, chambers, posts, andarrangement of posts, and paragraphs [0127] to [0130] which describe themethods of making the device are incorporated by reference herein.

In some embodiments, the method for identifying a compound for treatingcancer, comprises culturing a first and second aliquot of tumor cellspheroids in a three-dimensional microfluidic device as described hereinin the presence and absence of one or more test compounds, detecting achange in the ratio of live cells to dead cells in the aliquots of tumorcell spheroids.

In some embodiments, the change in the ratio of live cells to dead cellsin the aliquots of tumor cell spheroids is monitored by measuring thetotal fluorescence emitted by each of the first and second fluorophoredyes. Accordingly, in some embodiments, the dead cell fluorescence andthe live cell fluorescence can be added together to yield a live anddead cell total. In some embodiments, the amount of dead cells may beexpressed as a percentage of the dead cell fluorescence to the totalfluorescence. In some embodiments, the amount of live cells can beexpressed as a percentage of the live cell fluorescence to the total. Insome embodiments, the dead cell fluorescence and the live cellfluorescence can be expressed as a ratio. For example, in someembodiments, the ratio is expressed as dead cells/live cells or livecells/dead cells. In this manner, expressing as a ratio provides astandardized method of evaluating tumor cell spheroids.

In some embodiments, proliferation and/or dispersion of the tumor cellspheroids are also assessed.

In some embodiments, the method for identifying a compound orcombination of compounds for treating cancer, comprises obtaining tumorspheroids from an enzyme treated tumor sample, suspending a firstaliquot of the tumor spheroids in biocompatible gel, and suspending asecond aliquot of the tumor spheroids in biocompatible gel. In someembodiments, the method further comprises placing the first aliquot ofthe tumor spheroids in biocompatible gel in a first three-dimensionaldevice, and contacting the first aliquot with a first fluorophore dyeselective for dead cells and a second fluorophore dye selective for livecells, where the first fluorophore dye emits fluorescence at a firstwavelength when bound to a dead cell and the second fluorophore dyeemits fluorescence at a second wavelength different from the firstwavelength when bound to a live cell. In some embodiments, the methodfurther comprises measuring total fluorescence emitted by each of thefirst and second fluorophore dyes in the first aliquot. In someembodiments, the method further comprises culturing the second aliquotin a second three-dimensional device, contacting the second aliquot withthe first fluorophore dye and the second fluorophore dye, wherein thecontacting of the second aliquot with the first fluorophore dye andsecond fluorophore dye is carried out at least 24 hours after thecontacting of the first aliquot with the first fluorophore dye andsecond fluorophore dye. In some embodiments, the method furthercomprises measuring total fluorescence emitted by each of the first andsecond fluorophore dyes in the second aliquot, wherein an increase ordecrease in the ratio of live cells to dead cells in each of thealiquots may be assessed.

“Culturing patient-derived tumor cell spheroids in the presence of thetest compound” comprises introducing the test compound into the one ormore fluid channels of the device described herein, wherein the one ormore gel cage regions of the device comprises a gel in which the tumorspheroids are embedded; and culturing the tumor spheroids under suitableculture conditions. Suitable conditions include growing the tumor cellspheroids under standard cell culture conditions (e.g. at 37° C. in ahumidified atmosphere of >80% relative humidity air and 5 to 10% CO₂).

In some embodiments, techniques provided herein relate to evaluating theeffects of one or more therapeutic treatments in a tumor cell spheroidculture. In some embodiments, changes in the tumor cell spheroid cultureare evaluated using one or more luminescent probes (e.g., one or morefluorescent dyes). For example, in some embodiments, methods describedherein comprise steps of contacting a tumor cell spheroid culture with afirst fluorophore dye and a second fluorophore dye that each selectivelybind to a cell type that is different from the other, and where thefluorophore dyes emit fluorescence at different wavelengths when boundto the respective cell types. In some embodiments, the first fluorophoredye is selective for dead cells and the second fluorophore dye isselective for live cells. In some embodiments, the first fluorophore dyeis selective for live cells and the second fluorophore dye is selectivefor dead cells.

In some embodiments, one or more fluorophore dye (e.g., one or more of afirst fluorophore dye, one or more of a second fluorophore dye) isintroduced into the one or more fluid channels of the cell culturedevice. The techniques provided in the present disclosure are envisionedto be compatible with any suitable fluorophore dye (e.g.,fluorophore-containing dyes, stains, fluorescent labels, etc.). Suitablefluorophore dyes are known in the art and can be selected by apractitioner in view of a desired application of the techniquesdescribed herein. For example, in some embodiments, the one or morefluorophore dyes utilized in the techniques described herein can becompatible with staining under fixed or non-fixed conditions.

Fluorophore dyes that can be used for the detection of dead cells innon-fixed conditions include, by way of example and not limitation,DNA-dependent stains such as propidium iodide, DRAQ7, and 7-AAD.Fluorophore dyes that can be used for the detection of dead cells ineither fixed or non-fixed conditions include, by way of example and notlimitation, dyes listed in Table 1.

TABLE 1 Examples of dyes to stain for dead cells Excitation Emission Dye(max) (max) Propidium iodide 540 nm 620 nm DRAQ7 600 nm 697 nm 7-AAD 550nm 645 nm eBioscience Fixable Viability Dye eFluor ® 350 nm 455 nm 455UVeBioscience Fixable Viability Dye eFluor ® 450 405 nm 450 nm eBioscienceFixable Viability Dye eFluor ® 506 405 nm 506 nm eBioscience FixableViability Dye eFluor ® 520 488 nm 522 nm eBioscience Fixable ViabilityDye eFluor ® 660 633 nm 660 nm eBioscience Fixable Viability DyeeFluor ® 780 633 nm 780 nm BioLegend Zombie Aqua ™ 382 nm 510 nmBioLegend Zombie NIR ™ 718 nm 746 nm BioLegend Zombie Red ™ 600 um 624nm BioLegend Zombie Violet ™ 400 nm 423 nm BioLegend Zombie UV ™ 362 nm459 nm BioLegend Zombie Yellow ™ 396 nm 572 nm

The techniques provided in the present disclosure are envisioned to becompatible with any suitable fluorophore dye selective for live cellsknown in the art. Fluorophore dyes that can be used for the detection oflive or fixed cells include, by way of example and not limitation,DNA-dependent stains such as acridine orange, nuclear green LCS1(ab138904), DRAQ5 (ab108410), CyTRAK Orange, NUCLEAR-ID Red DNA stain(ENZ-52406), and SiR700-DNA. Examples of non-DNA-dependent fluorophoredyes that stain for live cells are known in the art and include, by wayof example and not limitation, calcein AM, calcein violet AM, andcalcein blue AM. Fluorophore dyes that can be used for the detection oflive or fixed cells include, by way of example and not limitation, dyesshown in Table 2.

TABLE 2 Examples of dyes to stain for live cells Dye Excitation EmissionVybrant ® DyeCycle ™ Violet UV, 405 nm 437 Vybrant ® DyeCycle ™ Green488 nm 534 Vybrant ® DyeCycle ™ Orange 488, 532 nm 563 Vybrant ®DyeCycle ™ Ruby 488, 633/5 nm 686 acridine orange 500 nm 526 nm nucleargreen LCS1 (ab138904) 503 nm 526 nm DRAQ5 (ab108410) 647 nm 681 nmCyTRAK Orange 488-550 nm 610 nm NUCLEAR-ID Red DNA stain 566 nm 650 nm(ENZ-52406) SiR700-DNA 689 nm 716 nm calcein AM 495 nm 515 nm calceinviolet AM 408 nm 450 nm calcein blue AM 360 nm 445 nm

Additionally, two-color cell viability assays are available and may beused with the techniques described herein. Examples of live/deadcytotoxicity kits include, by way of example and not limitation, kitsshown in Table 3.

TABLE 3 Examples of kits containing dyes to stain for both live/deadcells Fluorescent Ex/Em Em Kit Name Platform Dyes (nm) Colors LIVE/DEADFC, FM, calcein AM 494/517 Green Viability/Cytotoxicity Kit M ethidium517/617 (Live) for mammalian cells homodimer- Red 1 (Dead) LIVE/DEADCell- FC, FM, DiOC₁₈(3) 484/501 Green Mediated Cytotoxicity M propidium536/617 (Live) Kit for animal cells, iodide Red 2000 assays (Dead)LIVE/DEAD Sperm FC, FM SYBR ™ 14 485/517 Green Viability Kit 200-1,000dye 536/617 (Live) assays propidium Red iodide (Dead) LIVE/DEAD CellVitality FC, FM, SYTOX ™ 488/530 Green Assay Kit C₁₂- M Green dye488/575 (Dead) resazurin/SYTOX ™ C12- Red (Live) Green 1,000 assaysresazurin

Changes in the tumor cell spheroid culture which predict or demonstratea reduction in proliferation and/or dispersion of the tumor cellspheroids in the presence or absence of the test compound can bedetected using known methods in the art, such as, chemical or physicalmethods, or a combination thereof.

As described herein, a tumor cell spheroid sample is evaluated byanalyzing the relative amounts of live cells and dead cells in separatealiquots of the sample. In some embodiments, the relative amounts oflive cells and dead cells in an aliquot is determined by fluorescencemeasurement. For example, in some embodiments, fluorescence measurementcomprises total fluorescence acquisition of the emissions detected froma first fluorophore dye that selectively binds dead cells and a secondfluorophore dye that selectively binds live cells. Based on suchmeasurements, the relative emission levels can be parsed out to providethe amount of live cells relative to the amount of dead cells in thealiquot.

In some embodiments, the relative amounts or ratio of live cells to deadcells in an aliquot can be expressed as a percentage of the totalfluorescence, such that:

[(% live cells)+(% dead cells)=100%]

In some embodiments, the relative levels of live cells and dead cells ina first aliquot is compared to a similar measurement obtained for asecond aliquot that has been treated with a test compound. An increasein (% dead cells) in the presence of the test compound compared to thefirst aliquot is indicative that the test compound is effective forinducing cell death. In some embodiments, the percentage of dead cellsin the presence of an effective test compound is increased relative tothe absence of the test compound by about 5%, by about 10%, by about15%, by about 20%, by about 25%, by about 30%, by about 35%, by about40%, by about 50%, by about 60%, by about 70%, by about 80%, by about90%, or greater.

In some embodiments, a change in the tumor cell spheroid culture canfurther be detected visually, e.g., using confocal microscopy imaging.The images obtained can be analyzed as described in Aref et al. IntegrBiol (Camb). 2013 February; 5(2):381-9, the disclosure of which isincorporated by reference in its entirety. In particular, the paragraphson pages 387-388 relating to image acquisition and analysis (normalizeddispersion, Δ/Δ0, and normalized cell number (N/N0)) are incorporated byreference in their entirety.

In some embodiments, the change in the tumor cell spheroid culture is aclustering of immune cells around one or more tumor cell spheroids inthe culture. In some embodiments, the change in the tumor cell spheroidculture is a decrease in size and/or number of cells of one or moretumor cell spheroids in the culture.

In some embodiments, the change in the tumor cell culture is furtherdetected chemically. For example, in some embodiments, the change in thetumor cell spheroid culture is determined by detection of the presenceof a biological molecule secreted into the culture supernatant. In someembodiments, the biological molecule is a protein, carbohydrate, lipid,nucleic acid, metabolite, or a combination thereof. In some embodiments,the biological molecule is a chemokine or a cytokine. In someembodiments, the cytokine is a growth factor (see e.g., Example 2). Insome embodiments, the biological molecule is known to be associated withactivation of the immune system or otherwise an enhancement of theimmune response.

In some embodiments, the detected biological molecule(s) involves singlecell sequencing of T cell receptors on tumor spheroid associated CD4 andCD8 T cells that become activated in the device.

In some embodiments, a sample of tumor cell spheroid culture supernatantmay be obtained from the 3D culture device. In some embodiments, asecreted biological molecule or a profile of secreted biologicalmolecules, e.g., a cytokine profile or chemokine profile, may bedetected in the tumor cell spheroid culture supernatant.

Methods for detecting secreted biological molecules are known in theart. Exemplary assays and cytokines are disclosed, for example, in WO2016/112172, the disclosure of which is incorporated herein byreference.

In some embodiments, a sample of tumor cell spheroid is analyzed bynucleic acid content. For example, it is possible to analyzeextracellular nucleic acids, e.g., nucleic acids present in a culturemedium of a tumor cell spheroid sample using methods known in the art.Alternatively, nucleic acids can be isolated from cells in a culturedsample using methods known in the art. In some embodiments, suchanalysis can be used to assess gene expression, e.g., to evaluate theexpression of genes associated with cytotoxicity, such as cytokines andcytokine receptors. In some embodiments, the cytokines are growthfactors (see e.g., Example 2). In some embodiments, DNA and/or RNA(e.g., mRNA, rRNA, tRNA, etc.) from a sample may be analyzed.

In some embodiments, RNA content of a sample is analyzed using RNAsequencing (RNA-seq). RNA-seq is a reliable, scalable, and comprehensivemethod to evaluate transcriptomic changes in the tumor immunemicroenvironment. The use of RNA-seq enables a broad and comprehensiveanalysis of changes in the tumor immune microenvironment associated withresponse and resistance to immune checkpoint blockade and furthermorecan readily be applied to molecular targeted therapies, combinationtherapies, engineered immune cells, and other related applications. Insome embodiments, RNA content is analyzed using single-cell RNA-seqmethodologies. In some embodiments, analysis by nucleic acid contentwill be utilized for medium- and high-throughput assay development.

In some embodiments, the test compound inhibits epithelial-mesenchymaltransition (EMT). In some embodiments, the test compound is a smallmolecule compound. In some embodiments, the methods described herein areused to screen a library of test compounds, for example, a library ofchemical compounds. In some embodiments, the test compound comprises anucleic acid molecule, for example, a DNA molecule, an RNA molecule, ora DNA/RNA hybrid molecule, single-stranded, or double-stranded. In someembodiments, the test compound comprises an RNAi compound, for example,an antisense-RNA, an siRNA, an shRNA, a snoRNA, a microRNA (miRNA), or asmall temporal RNA (stRNA). In some embodiments, the test compoundcomprises an aptamer. In some embodiments, the test compound comprises aprotein or peptide. In some embodiments, the test compound comprises anantibody or an antigen-binding antibody fragment, e.g., a F(ab′)2fragment, a Fab fragment, a Fab′ fragment, or an scFv fragment. In someembodiments, the antibody is a single domain antibody. In someembodiments, the compound comprises a ligand- or receptor-bindingprotein. In some embodiments, the compound comprises a gene therapyvector.

In some embodiments, more primary tumor cell spheroids are cultured inthe presence of more than one compound, e.g., a first test compound anda second test compound, optionally a third test compound, fourthcompound, etc.

In some embodiments, a test compound is an anti-cancer compound. In someembodiments a test compound is a chemotherapeutic compound, animmunomodulatory compound, or radiation.

Exemplary chemotherapeutic compounds include asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc. In some embodiments, a test compound is avinca alkaloid, e.g., vinblastine, vincristine, vindesine, vinorelbine.In some embodiments, a test compound is an alkylating compound. e.g.,cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide. Insome embodiments, a test compound is an antimetabolite, e.g., folic acidantagonists, pyrimidine analogs, purine analogs or adenosine deaminaseinhibitor, e.g., fludarabine. In some embodiments, a test compound is anmTOR inhibitor. In some embodiments, a test compound is a proteasomeinhibitor, e.g., aclacinomycin A, gliotoxin or bortezomib.

In some embodiments, a test compound is a small molecule inhibitor,e.g., a TBK1 inhibitor, a MEK inhibitor, a FAK inhibitor, a BRD/BETinhibitor, a CDK 4/6 inhibitor, an HDAC inhibitor, a DNMT inhibitor (orhypomethylating compound), a MET inhibitor, an EGFR inhibitor, or a BRAFinhibitor. In some embodiments, a test compound is a kinase inhibitor,e.g., a TBK1 inhibitor, a MEK inhibitor, a FAK inhibitor, or a CDK 4/6inhibitor. For example, TBK-1 inhibitors are known in the art, e.g., Yu,T., et al. “TBK1 inhibitors: a review of patent literature (2011-2014)”Exp. Opin. On Ther. Patents 25(12); Hasan, M., et al. J. Immunol.195(10):4573-77; and US20150344473, the contents of each of which areincorporated herein by reference.

Exemplary immunomodulatory compounds include immune activating compoundsor inhibitors of an immune checkpoint protein selected from the groupconsisting of: CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3 (CD276), B7-H4,4-1BB (CD137), OX40, ICOS, CD27, CD28, PD-L2, CD80, CD86, B7RP1, HVEM,BTLA. CD137L. OX40L, CD70, CD40, CD40L, GAL9, A2aR, and VISTA. In someembodiments, the immune checkpoint inhibitor is a peptide, antibody,interfering RNA, or small molecule. In some embodiments, the immunecheckpoint inhibitor, e.g., inhibitor, is a monoclonal antibody, or anIg fusion protein. In some embodiments, the immune checkpoint inhibitoris an antibody or an antigen binding fragment thereof. In someembodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.

In some embodiments, the immune checkpoint inhibitor inhibits PD. Inhumans, programmed cell death protein 1 (PD-1) is encoded by the PDCD1gene. PDCD1 has also been designated as CD279 (cluster ofdifferentiation 279). This gene encodes a cell surface membrane proteinof the immunoglobulin superfamily. PD-1 is a 288 amino acid cell surfaceprotein molecule. PD-1 has two ligands, PDL1 and PD-L2, which aremembers of the B7 family. PD-1 is expressed on the surface of activatedT cells. B cells, and macrophages. PD-1 is expressed in pro-B cells andis thought to play a role in their differentiation. See T. Shinohara 5et al., Genomics 23 (3): 704-6 (1995). PD-1 is a member of the extendedCD28/CTLA-4 family of T cell regulators. (Y. Ishida et al., “EMBO J. 1(11): 3887-95, (1992)). PD-1 may negatively regulate immune responses.PD1 limits autoimmunity and the activity of T cells in peripheraltissues at the time of an inflammatory response to infection.

An immune checkpoint inhibitor that inhibits PD-1, or a PD-1 antagonist,as used herein is a molecule that binds to PD-1 and inhibits or preventsPD-1 activation. Without wishing to be bound by theory, it is believedthat such molecules block the interaction of PD-1 with its ligand(s)PD-L1 and/or PD-L2.

PD-1 activity may be interfered with by antibodies that bind selectivelyto and block the activity of PD-1. The activity of PD-1 can also beinhibited or blocked by molecules other than antibodies that bind PD-1.Such molecules can be small molecules or can be peptide mimetics ofPD-L1 and PD-L2 that bind PD-1 but do not activate PD-1. Molecules thatantagonize PD-1 activity include those described in U.S. Publications20130280265, 20130237580, 20130230514, 20130109843, 20130108651,20130017199, and 20120251537, 2011/0271358, EP 2170959B1, the entiredisclosures of which are incorporated herein by reference. See also M.A. Curran, et al., Proc. Natl. Acad. Sci. USA 107, 4275 (2010); S. L.Topalian, et al., New Engl. J. Med. 366, 2443 (2012); J. R. Brahmer, etal., New Engl. J. Med. 366, 2455 (2012); and D. E. Dolan et al., CancerControl 21, 3 (2014). Herein, PD-1 antagonists include: nivolumab, alsoknown as BMS-936558 (Bristol-Meyers Squibb, and also known as MDX-1106or ONO-4538), a fully human IgG4 monoclonal antibody against PD-1:pidilizumab, also known as CT-011 (CureTech), a humanized IgG1monoclonal antibody that binds PD-1; MK-3475 (Merck, and also known asSCH 900475), an anti-PD-1 antibody; and pembrolizumab (Merck, also knownas MK-3475, lambrolizumab, or Keytruda), a humanized IgG4-kappamonoclonal antibody with activity against PD-1. Compounds that interferebind to the DNA or mRNA encoding PD-1 also can act as PD-1 inhibitors.Examples include a small inhibitory anti-PD-1 RNAi, an anti-PD-1antisense RNA, or a dominant negative protein. PD-L2 fusion proteinAMP-224 (codeveloped by Glaxo Smith Kline and Amplimmune) is believed tobind to and block PD-1.

In some embodiments, the immune checkpoint inhibitor inhibits PD-L1. Inhumans, programmed death-ligand 1 (PD-L1), also known as B7 homolog 1(B7-H1) or cluster of differentiation 274 (CD274), is a 40 kDa type 1transmembrane protein that is encoded by the CD274 gene. Foreignantigens normally induce an immune response triggering proliferation ofantigen-specific T cells, such as antigen-specific CD8+ T cells. PD-L1is an immune checkpoint inhibitor that may block or lower such an immuneresponse. PD-L1 may play a major role in suppressing the immune systemduring events such as pregnancy, tissue allografts, autoimmune disease,and other disease states, such as hepatitis and cancer. The PD-L1 ligandbinds to its receptor, PD-1, found on activated T cells, B cells, andmyeloid cells, thereby modulating activation or inhibition. In additionto PD-1. PD-L1 also has an affinity for the costimulatory molecule CD80(B7-1). Upon IFN-γ stimulation, PDL1 is expressed on T cells, naturalkiller (NK) cells, macrophages, myeloid dendritic cells (DCs). B cells,epithelial cells, and vascular endothelial cells.

PD-L1 activity may be blocked by molecules that selectively bind to andblock the activity of PD-L1. Anti-PD-L1 antibodies block interactionsbetween PD-L1 and both PD-1 and B7-1 (also known as CD80). Block meansinhibit or prevent the transmission of an inhibitory signal mediated viaPD-L1. PD-L1 antagonists include, for example: BMS-30 936559, also knownas MDX-1105 (Bristol-Meyers Squibb), a fully human, high affinity,immunoglobulin (Ig) G4 monoclonal antibody to PD-L1; MPDL3280A, alsoknown as RG7446 or atezolizumab (Genentech/Roche), an engineered humanmonoclonal antibody targeting PDL1; MSB0010718C, also known as avelumab(Merck), a fully human IgG1 monoclonal antibody that binds to PD-L1; andMEDI473 (AstraZeneca/MedImmune), a human immunoglobulin (Ig) G1κmonoclonal antibody that blocks PD-L1 binding to its 5 receptors.Compounds that bind to the DNA or mRNA encoding PD-L1 also can act asPD-L1 inhibitors, e.g., small inhibitory anti-PDL1 RNAi, smallinhibitory anti-PD-L1 RNA, anti-PD-L1 antisense RNA, or dominantnegative PD-L1 protein. Antagonists of or compounds that antagonizePD-L1, e.g., anti-PD-L1 antibodies and PD-L1 antagonists, may include,but are not limited to those previously mentioned and any of those thatare disclosed in Stewart et al., 2015, 3(9):1052-62; Herbst et al.,2014, Nature 515:563-567: Brahmer et al., N Engl J Med 2012:366:2455-2465; U.S. Pat. No. 8,168,179; US20150320859: and/orUS20130309250, all incorporated herein by reference.

In some embodiments, the immune checkpoint inhibitor inhibits CTLA-4.CTLA-4 (also known as CTLA-4 or cluster of differentiation 152 (CD152)),is a transmembrane glycoprotein that, in humans, is encoded by theCTLA-4 gene. CTLA-5 4 is a member of the immunoglobulin superfamily,which is expressed on the surface of helper T cells and is present inregulatory T cells, where it may be important for immune function.CTLA-4, like the homologous CD28, binds to B7 molecules, particularlyCD80/B7-1 and CD86/B7-2 on antigen-presenting cells (APCs), therebysending an inhibitory signal to T cells. CTLA-4 functions as an immunecheckpoint that inhibits the immune system and is important formaintenance of immune tolerance.

CTLA-4 activity may be blocked by molecules that bind selectively to andblock the activity of CTLA-4 or that bind selectively to itscounter-receptors, e.g., CD80, CD86, etc., and block activity of CTLA-4.Blocking means inhibit or prevent the transmission of an inhibitorysignal via CTLA-4. CTLA-4 antagonists include, for example, inhibitoryantibodies directed to CD80, CD86, and/or CTLA-4; small moleculeinhibitors of CD80, CD86, and CTLA-4; antisense molecules directedagainst CD80, CD86, and/or CTLA-4; adnectins directed against CD80,CD86, and/or CTLA-4; and RNAi inhibitors (both single and doublestranded) of CD80, CD86, and/or CTLA-4.

Suitable CTLA-4 antagonists and/or anti-CTLA-4 antibodies includehumanized anti-CTLA-4 antibodies, such as MDX-010/ipilimumab(Bristol-Meyers Squibb), tremelimumab/CP-675,206 (Pfizer; AstraZeneca),and antibodies that are disclosed in PCT Publication No. WO 2001/014424,PCT Publication No. WO 2004/035607, U.S. Publication No. 2005/0201994,European Patent No. EP 1212422 B1, U.S. Pat. Nos. 5,811,097, 5,855,887,6,051,227. 6,984,720, 7,034,121, 8,475,790, U.S. Publication Nos.2002/0039581 and/or 2002/086014, the entire disclosures of which areincorporated herein by reference. Other anti-CTLA-4 antibodies and/orCTLA-4 antagonists that can be used in a method of the presentdisclosure include, for example, those disclosed in Hurwitz et al.,Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al.,J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (antibodyCP9675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and Lipsonand Drake, Clin Cancer Res; 17(22) Nov. 15, 2011; U.S. Pat. No.8,318,916; and/or EP1212422B1, all of which are herein incorporated byreference, in their entireties.

In some embodiments, the immune checkpoint inhibitor inhibits VISTA.V-domain Ig suppressor of T cell activation (VISTA), (also known asPD-H1, PD-1 homolog, or Dies1), is a negative regulator of T cellfunction. VISTA is a 309 aa type I transmembrane protein that iscomposed of seven exons, it has one Ig-V like domain, and its sequenceis similar to the Ig-V domains of members of CD28 and B7 families. VISTAis highly expressed in the tumor microenvironment (TME) and onhematopoietic cells. It is also expressed on macrophages, dendriticcells, neutrophils, natural killer cells, and naive and activated Tcells. Its expression is highly regulated on myeloid antigen-presentingcells (APCs) and T cells, while lower levels are found on CD4+ T cells,CD8+ T cells, and Treg cells. VISTA shows some sequence homology to thePD-1 ligand, PD-L1, however the two immune checkpoint inhibitors arestructurally different and have different signaling pathways. VISTAblockade has been shown to enhance antitumor immune responses in mice,while in humans, blockade of the related PD-1 pathway has shown greatpotential in clinical immunotherapy trials. VISTA is a negativecheckpoint regulator that suppresses T-cell activation and its blockademay be an efficacious immunotherapeutic strategy for human cancer. VISTA(Wang et al., 2011. JEM. 208(3):577-92.; Lines et al., 2014. Cancer Res.74(7):1924-32.: Kondo et al. 2015. J. of Immuno.V194.; WO2011120013;US20140105912; US20140220012; US20130177557, US20130177557, incorporatedby reference herein, in their entireties).

VISTA activity may be blocked by molecules that selectively bind to andblock the activity of VISTA. Molecules or compounds that are VISTAantagonists include peptides that bind VISTA, antisense moleculesdirected against VISTA, single- or doublestranded RNAi moleculestargeted to degrade or inhibit VISTA, small molecule inhibitors ofVISTA, anti-VISTA antibodies, inhibitory antibodies directed to VISTA,and humanized antibodies that selectively bind and inhibit VISTA.Antagonists of or compounds that antagonize VISTA, e.g., anti-VISTAantibodies and VISTA antagonists, are not limited to, but may includeany of those that are disclosed in Liu et al. 2015. PNAS.112(21):6682-6687; Wang et al., 2011. JEM. 208(3):577-92; Lines et al.,2014. Cancer Res. 74(7):1924-32; Kondo et al. 2015. J, of Immuno.V194;WO2015097536, EP2552947. WO2011120013, US20140056892, U.S. Pat. No.8,236,304, WO2014039983, US20140105912, US20140220012, US20130177557;WO2015191881; US20140341920; CN105246507; and/or US20130177557, all ofwhich are incorporated by reference herein, in their entireties.

In some embodiments, the immune checkpoint inhibitor inhibits TIM-3. Insome embodiments, the immune checkpoint inhibitor inhibits LAG-3.

In some embodiments, a combination of test compounds are tested in thecell culture. In some embodiments, the combination of immune checkpointinhibitors includes a PD1 inhibitor and a CTLA-4 inhibitor. In someembodiments, the combination of immune checkpoint inhibitors includes aPD1 inhibitor and a TIM-3 inhibitor. In some embodiments, thecombination of immune checkpoint inhibitors includes a PD1 inhibitor anda LAG-3 inhibitor. In some embodiments, the combination of immunecheckpoint inhibitors includes a PD-L1 inhibitor and a CTLA-4 inhibitor.In some embodiments, the combination of immune checkpoint inhibitorsincludes a PD-L1 inhibitor and a TIM-3 inhibitor. In some embodiments,the combination of immune checkpoint inhibitors includes a PD-L1inhibitor and a LAG-3 inhibitor. In some embodiments, the combination ofimmune checkpoint inhibitors includes a CTLA-4 inhibitor and a TIM-3inhibitor. In some embodiments, the combination of immune checkpointinhibitors includes a CTLA-4 inhibitor and a LAG-3 inhibitor. In someembodiments, the combination of immune checkpoint inhibitors includes aTIM-3 inhibitor and a LAG-3 inhibitor. In some embodiments, thecombination of immune checkpoint inhibitors includes a PD1 inhibitor, aCTLA-4 inhibitor, and a TIM-3 inhibitor. In some embodiments, thecombination of immune checkpoint inhibitors includes a PD1 inhibitor, aCTLA-4 inhibitor, and a LAG-3 inhibitor. In some embodiments, thecombination of immune checkpoint inhibitors includes a PD1 inhibitor, aTIM-3 inhibitor, and a LAG-3 inhibitor. In some embodiments, thecombination of immune checkpoint inhibitors includes a CTLA-4 inhibitor,a TIM-3 inhibitor, and a LAG-3 inhibitor.

In some embodiments, the combination of test compounds includes animmune checkpoint inhibitor and a small molecule compound. In someembodiments, the combination of test compounds includes an immunecheckpoint inhibitor and a TBK-1 inhibitor. In some embodiments, thecombination of test compounds includes a PD-1 inhibitor and a TBK-1inhibitor. In some embodiments, the combination of test compoundsincludes a PD-L1 inhibitor and a TBK-1 inhibitor. In some embodiments,the combination of test compounds includes a CTLA-4 inhibitor and aTBK-1 inhibitor. In some embodiments, the combination of test compoundsincludes a VISTA inhibitor and a TBK-1 inhibitor. In some embodiments,the combination of test compounds includes a TIM-3 inhibitor and a TBK-1inhibitor. In some embodiments, the combination of test compoundsincludes a LAG-3 inhibitor and a TBK-1 inhibitor.

In some embodiments, an immune activating compound is a CD28 antagonist,e.g., an anti-CD28 antibody. An antibody, or immunoglobulin, is aglycoprotein containing two identical light chains (L chains), eachcontaining approximately 200 amino acids, and two identical heavy chains(H chains), which generally are at least twice as long as the L chains.The paratope of the antibody is specific for a particular epitope of anantigen, and their spacial complementarity (binding) “tags” the microbefor further action or neutralize its actions directly. The antibodycommunicates with other components of the immune response via itscrystallizable fragment (Fc) region, which includes a conservedglycosylation site. There are five Fc regions, resulting in the fivedifferent antibody isotypes: IgA, IgD, IgE, IgG, and IgM. IgD functionsas an antigen receptor on B cells that have not been exposed toantigens, and activates basophils and mast cells, resulting in theproduction of antimicrobial factors. IgG, expressed in four forms,provides the majority of antibody-based immunity against invadingpathogens. IgM is expressed on the surface of B cells as a monomer, andin a secreted form as a pentamer. It eliminates pathogens during theearly phases of humoral (B cell-mediated) immunity before there 5 aresufficient levels of IgG. IgG is often used in immunotherapy.

The term antibody is used in the broadest sense and specificallyincludes, for example, single monoclonal antibodies, antibodycompositions with polyepitopic specificity, single chain antibodies, andantigen-binding fragments of antibodies. An antibody may include animmunoglobulin constant domain from any immunoglobulin, such as IgG1,IgG2, IgG3, or IgG4 subtypes, IgA (including IgA 1 and IgA2), IgE, IgDor IgM.

A fluorescence light source provides an excitation wavelength to excitea fluorophore. A fluorescent light source can be used in connection witha photosensitive detector capable of detecting total fluorescenceemitted by the excited fluorophore to generate an image. A fluorescencemicroscope can be configured to include a fluorescence light source anda photosensitive detector. Fluorescence microscopes are available in theart and are commercially available. A skilled artisan would be able toselect a microscope with appropriate capabilities for the desiredimaging and/or analysis.

Fluorescence microscopes are available in upright or invertedconfigurations, either of which are suitable for use with the providedmethods. In an upright microscope configuration, the objective andphotosensitive detector, e.g., camera, is disposed above the stage,pointing down. Exemplary upright fluorescent microscopes include, butare not limited to. Nikon Eclipse 80i Fluorescent Microscope. In aninverted microscope configuration, the light source is disposed abovethe stage pointing down, and the objective is disposed below the stagepointing up.

In some embodiments, a fluorescence microscope is an epifluorescencemicroscope, in which light of the appropriate excitation wavelength ispassed through the objective lens onto the sample to be examined. Insome such embodiments, fluorescence is emitted from the sample throughthe same objective lens through which the light initially passed, and isthen detected via a photosensitive detector, e.g., a camera. In someinstances, a dichroic beam splitter may be used to filter the emittedfluorescence by permitting fluorescence of a particular wavelength topass through, while reflecting the remaining fluorescence, to increasethe signal-to-noise ratio. In some embodiments, a fluorescencemicroscope is a confocal microscope. Confocal microscopes can be used,for example, to generate three dimensional structures from a series ofoptical section images. In some embodiments, a fluorescence microscopeis a total internal reflection fluorescence microscope.

In some embodiments, the fluorescence microscope is configured to permitmeasurement of emission of fluorescence from two or more distinctfluorophore dyes at distinct wavelengths. For example, in someembodiments, the fluorescence microscope contains a filter arrangementthat allows separation of the fluorescence emitted by the two or moredistinct fluorophore dyes.

In some embodiments, the microscope is configured to permit measurementof multiple capture images of the same microfluidic device by moving thestage on which the microfluidic device is disposed. In some embodiments,the stage is a motorized stage. Motorized stages are known in the artand are commercially available. In some embodiments, the stage isconfigured to move one dimensionally, e.g., to move in the x-axis. Insome embodiments, the stage is configured to move two dimensionally,e.g., to move in the x- and y-axes. In some embodiments, the stage isconfigured not to move in three dimensions, e.g., to not move in thez-axis. In some such embodiments, the focal plane of the tumor cellspheroids in the three-dimensional microfluidic device remains the samethroughout the fluorescence detection.

In some embodiments, a three dimensional analysis of the tumor cellspheroids is performed. For example, in some embodiments, the stage isconfigured to move in three dimensions, e.g., moves in x-, y-, andz-axes. In such embodiments, images of multiple sections (e.g., focalplanes) can be obtained to generate a Z stack using microscopes,photosensitive detectors, and software available in the art. Forexample, commercially available ProScan controller and TTL Breakout Box(Prior Scientific, Rockland, Mass.) can be used to control a CCD camerafor Z-stack image acquisition.

The overall resolution of the fluorescence emission detection depends onthe magnification of the objective lens through which the detectablesignal passes, and/or the magnification of the photosensitive detectiondevice, e.g., camera. In some embodiments, the magnification of theobjective lens is at least 2×, at least 3×, at least 4×, at least 10×,at least 40×, at least 100×, or more. In some embodiments, themagnification of the photosensitive detection device, e.g., camera, is0×. In some embodiments, the magnification of the photosensitivedetection device. e.g., camera, is at least 2×, at least 3×, at least4×, at least 10×, or more. In some embodiments, the overall resolutionof the fluorescence detection is at least 2×, at least 3×, at least 4×,at least 10×, at least 40×, at least 100×, at least 400×, at least1000×, or more. In some embodiments, the resolution at which thefluorescence is detected is such that the entire field of the threedimensional device containing the tumor cell spheroids can be imagedwithout adjusting the focal plane.

Images of the fluorescence emitted by the fluorophore dyes are detectedand/or captured using a photosensitive detector. Photosensitivedetectors capable of detecting total fluorescence are known in the artand commercially available. In some embodiments, a photosensitivedetector is a camera or a photodiode. In some embodiments, the camera isa CCD camera. Exemplary cameras include, but are not limited to CoolSNAPCCD Camera (Roper Scientific). In some embodiments, the photodiode is anavalanche photodiode.

Images can be acquired by the photosensitive detector and analyzed,e.g., using commercially available software. Exemplary image capture andanalysis software includes, but is not limited to NIS-Elements ARsoftware package (Nikon). In some embodiments, multiple images of amicrofluidic device are captured by the detector and stitched together.Images may be deconvoluted using available programs such as AutoQuant(Media Cybernetics).

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1. Materials and Methods for Ex Vivo Profiling PatientSamples.

A cohort of patients (Tables S1-1 and S1-2) treated at MassachusettsGeneral Hospital and Dana-Farber Cancer Institute was assembled forPDOTS profiling and culture between August 2015 and August 2016.Informed consent was obtained from all subjects. Tumor samples werecollected and analyzed according to Dana-Farber/Harvard Cancer CenterIRB-approved protocols. These studies were conducted according to theDeclaration of Helsinki and approved by the MGH and DFCI IRBs.De-identified archival matched plasma and tissue samples from the MGHMelanoma Tissue Bank were obtained from patients pre- or on-treatmentwith anti-PD-1 therapy as indicated. Mutational analysis performed onclinically validated next-generation sequencing (NGS) platforms at MGHand DFCI. Clinical benefit (CB) to PD-1 was defined as absence ofdisease, decrease in tumor volume (radiographic or clinical), or stabledisease, whereas patients with no clinical benefit (NCB) includedpatients with mixed response, progression on treatment, and primarynon-responders.

TABLE S1-1 Mutational ID Age/Sex Diagnosis Stage Status MGH-01 78/MMelanoma IV BRAF^(WT), (cutaneous) NRAS^(WT) MGH-02 69/M Melanoma IIIBN/A (cutaneous) MGH-03 49/M Melanoma IV N/A (cutaneous) MGH-04 (**) 28/MMelanoma IV BRAF^(V600E) (cutaneous) MGH-05 70/M Melanoma IV BRAF^(WT),(cutaneous) NRAS^(G13V) MGH-06 78/M Melanoma IV BRAF^(WT), (cutaneous)NRAS^(WT) MGH-07 (**) 28/M Melanoma IV BRAF^(V600E) (cutaneous) MGH-0880/M Melanoma IV BRAF^(V600K) (cutaneous) MGH-09 69/F Melanoma IVBRAF^(V600E) (cutaneous) MGH-10 74/F Melanoma IV KIT^(D816A) (mucosal)MGH-11 63/F Melanoma IV N/A (cutaneous) MGH-12 (□) 61/M Melanoma IV TP53(cutaneous) MGH-13 65/F Thyroid cancer IV BRAF^(V600E) (papillary)MGH-14 (□) 61/M Melanoma IV TP53 (cutaneous) MGH-16 62/M Melanoma (sino-IV NRAS^(G12D) nasal) MGH-18 68/M Melanoma IV NRAS^(Q61R) (cutaneous)MGH-19 24/F Adreno-cortical II N/A carcinoma DFCI-01 73/F Melanoma IIINot tested (cutaneous) DFCI-02 (#) 81/M Melanoma IV CDKN2A (Q50*)(cutaneous) DFCI-04 72/F Mesothelioma IV 9p del, polysomy 22 DFCI-0642/F Merkel cell IV BRCA2 (F439V) carcinoma DFCI-07 58/M NSCLC/lung IVKRAS/LKB1 adeno-carcinoma DFCI-09 45/M Pancreatic IIB KRAS^(G12D), TP53adenocarcinoma DFCI-10 ({circumflex over ( )}) 55/M Thyroid cancer IVTP53, MSH2, (Hurthle cell) PTEN DFCI-12 58/F Head/neck IV Unknownsquamous cell carcinoma DFCI-13 (*) 76/M Thyroid cancer IV Unknown(papillary) DFCI-15 75/F Thyroid cancer IV BRAF^(V600E) (papillary)DFCI-16 (*) 76/M Thyroid cancer IV Unknown (papillary) DFCI-17 (#) 81/MMelanoma IV CDKN2A (Q50*) (cutaneous) DFCI-18 (*) 76/M Thyroid cancer IVUnknown (papillary) DFCI-19 67/M Pancreatic IIA Unknown adenocarcinomaDFCI-20 51/M Thyroid cancer IV None identified (papillary) DFCI-21 (*)76/M Thyroid cancer IV Unknown (papillary) DFCI-22 (*) 76/M Thyroidcancer IV Unknown (papillary) DFCI-24 79/M Small cell lung IV Unknowncancer DFCI-25 68/M Melanoma IV NRAS^(Q61K), (cutaneous) CDKN2A (Q50*)DFCI-27 72/M Melanoma IV BRAF^(WT) (cutaneous) DFCI-29 ({circumflex over( )}) 55/M Thyroid cancer IV TP53, MSH2, (Hurthle cell) PTEN DFCI-3052/M Merkel cell IV Unknown carcinoma DFCI-31 67/M Merkel cell IVUnknown carcinoma DFCI-32 89/M Merkel cell IIIb Unknown carcinomaDFCI-33 49/M Thyroid cancer IV Unknown (differentiated) (*) DFCI-13, 16,18, 21, 22 represent serial pleural effusion samples from same patient.(**) MGH-04 and MGH-07 are serial biopsies from the same patient. (#)DFCI-02 and -17 are samples from same patient. ({circumflex over ( )})DFCI-10 and DFCI-29 are samples from the same patient. (□) MGH-12 andMGH-14 are serial samples from a patient with a brain metastasisrequiring resection and subsequent re-resection.

TABLE S1-2 Therapy at Prior Time of Subsequent ID Therapy Biopsy SiteTherapy Response to Status MGH-01 None None subcutaneous None N/ADeceased; never treated MGH-02 None None lymph node None N/A Alive; NEDMGH-03 None None lymph node BRAFi + MEKi N/A Deceased; → αPD-1 +progression αCTLA-4 MGH-04 None None subcutaneous αPD-1 + N/A Alive;ongoing (**) αCTLA-4 → response αPD-1 maintenance MGH-05 αCTLA-4 → αPD-1→ None subcutaneous None Progression Deceased; clinical trialprogression MGH-06 αPD-1 αPD-1 lymph node αPD-1 (irAE) → Mixed Alive;ongoing αCTLA-4 response MGH-07 αPD-1 + αCTLA-4 αPD-1 + SubcutaneousαPD-1 Response Alive; ongoing (**) αCTLA-4 response MGH-08 αPD-1+ αPD-1Subcutaneous αCTLA-4 → Mixed Alive; BRAFi + MEKi Response ongoingresponse to BRAFi + MEKi MGH-09 αPD-1 → irAE None Subcutaneous BRAFi +Response Alive; MEKi (SD × 4-5 ongoing mo) → PD response to off-therapyBRAFi + MEKi MGH-10 Chemotherapy, radiation, None Small None ResponseAlive; NED surgery → αCTLA-4 → bowel (partial)— αPD-1 held for irAEMGH-11 BRAFi/MEKi None Lymph node αPD-1 Response Alive; ongoingtreatment MGH-12 αCTLA-4 (PD) → αPD-1 αPD-1 Brain αPD-1 Mixed Alive;mixed (□) (MR) metastasis Response response MGH-13 Radioactive iodineNone Subcutaneous Radiation N/A Alive; awaiting further therapy MGH-14αPD-1 αPD-1 Brain αPD-1 + Mixed Alive; stable (□) metastasis radiationResponse disease MGH-16 Anti-PD-L1 + MEKi (PD) → None Subcutaneous NoneProgression Deceased high-dose IL-2 (AE) → αCTLA-4 (PD) → αPD-1 (MR →PD) → SIRT (liver metastases) → pan-RAFi (PD) MGH-18 Radiation →anti-PD-1 + αPD-1 Subcutaneous αPD-1 Progression Alive; anti-KIR (irAE)→ (slow) slowly αCTLA-4 (PD) → pan-RAFi progressive (PD) → αPD-1 +radiation disease MGH-19 None None Primary None N/A Alive DFCI-01 NoneUnknown Lymph node N/A Unknown Unknown DFCI-02 (#) αCTLA-4, αPD-1 aPD-1Subcutaneous αPD-1 Progression Deceased DFCI-04 None Unknown PrimaryNone N/A Deceased (partial pleurectomy) DFCI-06 None None SubcutaneousNone N/A Deceased DFCI-07 Chemotherapy (PD) → αPD-1 None Peritonealfluid None Progression Deceased DFCI-09 None None Pancreas None N/AAlive; recurrent (Whipple) disease DFCI-10 ({circumflex over ( )}) NoneNone Subcutaneous lenvatinib N/A Alive DFCI-12 Cisplatin None Pleuralbiopsy αPD-1 Progression Deceased (VATS) DFCI-13 (*) MLN-0128 MLN-0128Pleural effusion MLN-0128 Progression Deceased DFCI-15 MLN-0128 MLN-0128Pleural effusion None N/A Deceased DFCI-16 (*) MLN-0128 None Pleuraleffusion nivolumab N/A Deceased DFCI-17 (#) RTA 408 + αCTLA-4 RTA 408 +Subcutaneous N/A Progression Alive; αCTLA-4 Progressive Disease DFCI-18(*) MLN-0128 None Pleural effusion nivolumab Progression DeceasedDFCI-19 FOLFIRINOX → None Pancreas 5-FU N/A Alive (NED) capecitabine +radiation (Whipple) DFCI-20 lenvatinib lenvatinib Pleural effusionMLN-0128 N/A Progressive Disease DFCI-21 (*) αPD-1 αPD-1 Pleuraleffusion αPD-1 Progression Deceased DFCI-22 (*) αPD-1 αPD-1 Pleuraleffusion αPD-1 Progression Deceased DFCI-24 cisplatin-etoposide → αPD-1αPD-1 Pleural effusion None Progression Deceased DFCI-25 αCTLA-4 + αPD-1→ None Lymph node N/A Response Alive; awaiting αPD-1maintenance → irAE(held for irAE) further therapy DFCI-27 αPD-1 αPD-1 Subcutaneous αPD-1Mixed Alive; on Response treatment DFCI-29 ({circumflex over ( )})Lenvatinib → MLN-0128 → everolimus Subcutaneous everolimus N/A Aliveeverolimus DFCI-30 carboplatin-etoposide, None Lymph node αPD-1 ResponseAlive; on radiation (recurrence) treatment DFCI-31 carboplatin-etoposideNone Subcutaneous αPD-1 Stable Disease Alive; on treatment DFCI-32 NoneNone Primary Radiation N/A Alive; on treatment DFCI-33 Radioactiveiodine None Pleural effusion lenvatinib N/A Alive; on treatment (*)DFCI-13, 16, 18, 21, 22 represent serial pleural effusion samples fromsame patient. (**) MGH-04 and MGH-07 are serial biopsies from the samepatient. (#) DFCI-02 and -17 are samples from same patient. ({circumflexover ( )}) DFCI-10 and DFCI-29 are samples from the same patient. (□)MGH-12 and MGH-14 are serial samples from a patient with a brainmetastasis requiring resection and subsequent re-resection. AE = adverseevent. irAE = immune-related adverse event. MR = mixed response. PD =progressive disease

Syngeneic Murine Models.

All animal experiments were performed in compliance with establishedethical regulations and were approved by the Dana-Farber Animal Care andUse Committee. MC38 murine colon adenocarcinoma cells were generouslyprovided by Dr. Gordon Freeman (DFCI) received under an MTA from Dr.Jeffrey Schlom of NCI (Bethesda, Md.). B16F10 melanoma cells, CT26 coloncarcinoma cells, and EMT6 breast mammary carcinoma cells were obtainedfrom ATCC. Cells were expanded, tested free for mycoplasma and mousepathogens. Thawed cells were cultured for up to three passages in DMEM(MC38, B16F10) or RPMI 1640 (CT26) supplemented with 10%heat-inactivated FBS at 37° C. in a humidified incubator maintained at5% CO₂. Cell counts were performed prior to implantation by bothhemocytometer & Invitrogen Countess Cell Counter. MC38 colon carcinomacells. CT26 colon carcinoma cells, and B16F10 melanoma cells (5×10⁵cells/mouse in 100 μL), re-suspended in sterile PBS (Ca⁺, Mg⁺ free),were injected into 8 week old female C57BL/6 albino mice or Balb/c(Jackson) and tumors were collected 2-3 weeks post-implantation or onreaching 2000 mm³ in size (or if there were any humane reason, includingdecreased BW >15% for 1 week or moribund) and MDOTS were prepared asdescribed below. Implantation of CT26 colon carcinoma cells wasperformed using BALB/c mice in identical fashion. For in vivo treatmentstudies, mice were randomized (using the deterministic method) and theninjected with 10 mg/kg isotype control IgG (clone 2A3, BioXCell) orrat-anti-mouse PD-1 (clone RMP1-14, BioXCell) every 3 days×8 doses andtumor volume was measured as shown. Investigators were not blinded totreatment groups. Tumor volume (TV) was monitored on a weekly basisafter the initial tumor volume is about 100 mm³. TV was measured twiceweekly during the exponential tumor growth phase, and body weight wasmonitored on a weekly basis after implantation.

Spheroid Preparation and Microfluidic Culture.

Fresh tumor specimens (murine and human patients) were received in media(DMEM) on ice and minced in a 10 cm dish (on ice) using sterile forcepsand scalpel. Minced tumor was resuspended in DMEM (4.5 mM glucose. 100mM sodium pyruvate, 1:100 penicillin-streptomycin) (Corning CellGro,Manassas, Va.)+10% FBS (Gemini Bio-Products, West Sacramento, Calif.),100 U/mL collagenase type IV (Life Technologies, Carlsbad. Calif.), and15 mM HEPES (Life Technologies, Carlsbad, Calif.), except for CT26tumors that were prepared in RPMI. Samples were pelleted and resuspendedin 10-20 mL media. Red blood cells were removed from visibly bloodysamples using RBC Lysis Buffer (Boston Bio-Products. Ashland. Mass.).Samples were pelleted and then resuspended in fresh DMEM+10% FBS andstrained over 100 μm filter and 40 μm filters to generate S1 (>100 μm),S2 (40-100 μm), and S3 (<40 μm) spheroid fractions, which weresubsequently maintained in ultra low-attachment tissue culture plates.S2 fractions were used for ex vivo culture. An aliquot of the S2fraction was pelleted and resuspended in type I rat tail collagen(Corning, Corning, N.Y.) at a concentration of 2.5 mg/mL followingaddition of 10×PBS with phenol red with pH adjusted using NaOH (pH7.0-7.5 confirmed using PANPEHA Whatman paper (Sigma-Aldrich, St. Louis,Mo.)). The spheroid-collagen mixture was then injected into the centergel region of the 3D microfluidic culture device. Collagen hydrogelscontaining PDOTS/MDOTS were hydrated with media with or withoutindicated therapeutic monoclonal antibodies after 30 minutes at 37° C.MDOTS were treated with isotype control IgG (10 μg/mL, clone 2A3) oranti-PD-1 (0.1, 1.0, 10 μg/mL, clone RMP1-14). Monoclonalrat-anti-mouse-CCL2 (5 μg/mL, clone 123616, R&D Systems) was used forCCL2 neutralization in MDOTS. PDOTS were treated with anti-PD-1(pembrolizumab, 250 μg/mL), anti-CTLA-4 (ipilimumab, 50 μg/mL), orcombination (250 μg/mL pembrolizumab+50 μg/mL ipilimumab). Doses wereselected (1:100 dilutions of stock concentrations used clinically) tocorrespond to reported peak plasma concentrations of each drug followingadministration of 10 mg/kg (FDA CDER application). In selectexperiments, PDOTS were treated with InVivoMAb human IgG isotype control(BioXCell). For spheroid cultures lacking immune cells, MC38 or CT26cells (1×10⁶) were seeded in low attachment conditions for 24 hours andwere filtered (as above). The S2 fraction was pelleted and resuspendedin collagen (as above) prior to microfluidic culture.

Flow Cytometric Immune Profiling of Murine Tumors and MDOTS.

Tumors from MC38 and B16F10 syngeneic murine models were procured asdescribed above. Cells were incubated for 20 minutes in the dark at roomtemperature using the Zombie NIR Fixable Viability Kit (Biolegend.423105) at a dilution of 1:500 in PBS. FcR were blocked by incubationwith the anti-mouse CD16/CD32 clone 2.402 blocking Ab (FisherScientific) for 15 minutes at 4° C., at a 1:100 dilution in flowcytometry staining buffer (PBS+5% FBS). Cell surface staining wasperformed by incubation for 20 minutes at 4° C. using the following Absdiluted in flow cytometry staining buffer (total staining volume of 100uL): Lymphocyte Staining Panel—CD45 AF488 (BioLegend 103122), CD25 PE(BioLegend 101904), CD19 PE-Dazzle (115554), CD49b PE-Cy7 (BioLegend,108922), CD3 BV421 (BioLegend, 100228), CD8 BV510 (BioLegend 100752).CD4 BV786 (Fisher Scientific #BDB563331); Myeloid Staining Panel—F4/80AF488 (BioLegend #123120), MHCII PE (BioLegend #107608). CD11c BV421(BioLegend #117330). Ly6G BV510 (Biolegend #127633), CD11b BV650(BioLegend #101239). Ly6C BV711 (BioLegend #128037), CD45 BV786 (FisherScientific #BDB564225), CD19 APC-Cy7 (BioLegend #115530), and CD49bAPC-Cy7 (BioLegend #108920) were included with the myeloid stainingpanel to be used as a dump channel along with the dead cells asdetermined by the Zombie NIR viability stain. After cell surfacestaining, cells were fixed by incubating in 200 μL IC Fixation Buffer(eBioscience #00-8222-49) for 10 minutes at room temperature. Cells werewashed and resuspended in flow cytometry staining buffer and read thefollowing day on a BD LSR Fortessa flow cytometer. Data were analyzedusing FlowJo software version 10.0.8.

Flow Cytometric Immune Profiling of PDOTS and Human Tumor Samples.

Cells were incubated with Live/Dead Fixable Yellow Dead Cell Stain Kit(Life Technologies, Carlsbad, Calif.) for 8 minutes in the dark at roomtemperature or Live/Dead Fixable Zombie NIR™ (Biolegend, San Diego,Calif.) for 5 minutes in the dark at room temperature in FACS buffer(PBS+2% FBS) at a ratio of 250 μL L/D 1× dilution per 100 mg of originalsample weight. Surface marker and intracellular staining were performedaccording to the manufacturer's protocol (eBioscience. San Diego,Calif.). FcR were blocked prior to surface antibody staining using HumanFcR Blocking Reagent (Miltenyi, San Diego, Calif.). Cells were fixed in1% PBS+2% FBS and washed prior to analysis on a BD LSRFortessa withFACSDiva software (BD Biosciences, San Jose, Calif.). Data were analyzedusing FlowJo (Ashland, Oreg.) software version 10.0.8. Cell viabilitywas determined by negative live/dead staining. Antibodies were specificfor the following human markers: CD3 (HIT3a: UCHT1), CD8 (RPA-T8), CD14(M5E2: MphiP9), CD45 (HI30). CD56 (B159), CCR7 (150503), EpCAM (EBA-1),HLA-DR (G46-6), PD-1 (EH12.1), and IgG1 isotype control (MOPC-21) fromBD Biosciences (San Jose, Calif.): CD3 (UCHT1), CD4 (RPA-T4), CD14(M5E2), CD15 (W6D3), CD16 (3G8), CD19 (HIB19), CD25 (BC96), CD33 (WM53),CD38 (HIT2), CD40L (24-31), CD45 (H130), CD45RA (HI100), CD45RO (UCHL1),CD56 (HCD56; 5.1H11), CD66b (G10F5), CD69 (FN50), CD123 (6H6), CD163(GHI/61), CTLA-4 (L3D10), CXCR5 (J252D4), EpCAM (9C4), Ki-67 (Ki-67),PD-1 (EH12.2H7), PD-L1 (29E.2A3), PD-L2 (24F.10C12), TIM-3 (F38-2E2),IgG2a isotype control (MOPC-173), IgG2b isotype control (MPC-11), andIgG1 isotype control (MOPC-21) from BioLegend (San Diego, Calif.);Pan-cytokeratin (C11) and PD-L1 (E L3N) from Cell Signaling Technologies(Danvers, Mass.); CD45 (2D1), FOXP3 (236A/E7), and IL-10 (236A/E7) fromAffymetrix/eBioscience (San Diego. Calif.). Four-way flow sorting ofimmune cells (CD45+), tumor cells (CD45−CD31−CD90−), cancer-associatedfibroblasts (CD45−CD31−CD90+), and endothelial cells (CD45−CD31+CD144+)was conducted on a BD Aria II SORP with gates set using single staincontrols and manual compensation using the following antibodies:CD31-APC (Biolegend, 303115), CD45-BV711 Biolegend, 304050). CD90-PE/Cy7(Biolegend, 328123), and CD144-PE (Biolegend, 348505). Cells were sortedinto cold PBS and stored on ice before mRNA extraction using establishedtechniques.

Microfluidic Device Design and Fabrication.

Microfluidic device design and fabrication was performed according tomethods known in the art (e.g., Aref, A. R. et al., Integr. Biol.(Camb.) (2013), the content of which is incorporated herein by referencein entirety), with modifications of device dimensions to accommodatelarger volumes of media. MDOTS were also evaluated using DAX-1 3D cellculture chip (AIM Biotech, Singapore), for select studies.

Live/Dead Staining.

Dual labeling was performed by loading microfluidic device with NexcelomViaStain™ AO/PI Staining Solution (Nexcelom, CS2-0106). Followingincubation with the dyes (20 minutes at room temperature in the dark),images were captured on a Nikon Eclipse 80i fluorescence microscopeequipped with Z-stack (Prior) and CoolSNAP CCD camera (RoperScientific). Image capture and analysis was performed using NIS-ElementsAR software package. Image deconvolution was done using AutoQuantModule. Whole device images were achieved by stitching in multiplecaptures. Live and dead cell quantitation was performed by measuringtotal cell area of each dye. Three different laboratories verifiedimmune-mediated cell death of MC38 MDOTS following PD-1 blockade. Toinhibit CD8+ T cell cytotoxicity, CT26 MDOTS were treated with 10 μg/mLanti-CD8a Ab (clone 53-6.72, BioXCell). Intertumoral and intratumoralheterogeneity experiments were performed using CT26 allografts asdescribed above. MDOTS were prepared using separate pieces of a largertumor alongside MDOTS prepared from a smaller allograft. MDOTS wereprocessed, treated, and profiled as described above. Immunofluorescencefor CD8+ T cells was performed as described below.

Immunofluorescence and Time-Lapse Imaging.

For immunofluorescence studies, PDOTS and MDOTS were washed with PBS andblocked with FcR blocking reagent (PDOTS—Miltenyi, Cambridge, Mass.,MDOTS—BioLegend, San Diego, Calif.) for 30 minutes at room temperature.Directly conjugated antibodies for PDOTS were CD326 EpCAM-PE (clone9C4), CD45-AlexaFluor-488 (HI30). CD8a-AlexaFluor488 (RPA-T8); forMDOTS—CD45-AlexaFluor488 or 647 (30-F11), CD8a-PE (53-6.7) (BioLegend,San Diego, Calif.). Antibodies were diluted 1:50 in 10 ug/mL solution ofHoechst 33342 (Thermo Fisher Scientific, Waltham, Mass.) in PBS andloaded into microfluidic devices for 1-hour incubation at room temp inthe dark. Spheroids were washed twice with PBS with 0.1% Tween20followed by PBS. For viability assessment, microfluidic devices wereloaded with 1:1000 solution of calcein AM (Thermo Fisher Scientific,Waltham, Mass.) in PBS. Images were captured on a Nikon Eclipse 80ifluorescence microscope equipped with Z-stack (Prior) and CoolSNAP CCDcamera (Roper Scientific). Image capture and analysis was performedusing NIS-Elements AR software package. Brightfield time-lapse imageswere captured with a 10×NA 0.3 objective and cooled CCD camera (Orca R2,Hamamatsu) in a humidified, temperature-controlled chamber. Illuminationwas with a CoolLED pe-100 white light LED. Time lapse imaging of severalfields of view over time was controlled by NISElements software of aPrior motorized stage along with the LED and camera.

Cytokine Profiling.

Two multiplex assays were performed utilizing a bead-based immunoassayapproach, the Bio-Plex Pro™ Human Cytokine 40-plex Assay (Cat#171AK99MR2) and Bio-Plex Pro™ Mouse Cytokine Panel 1, 23-plex (Cat#M60009RDPD) on a Bio-plex 200 system (Cat #171000201). MDOT/PDOTconditioned media concentration levels [μg/mL] of each protein werederived from 5-parameter curve fitting models. Fold changes relative tothe MDOT/PDOT control were calculated and plotted as log 2FC. Lower andupper limits of quantitation (LLOQ/ULOQ) were imputed from standardcurves for cytokines above or below detection. Conditioned media fromPDOTS were assayed neat and plasma was diluted 1:4.

Tertiary Lymphoid Structure Evaluation.

Fifty-two sections stained with hematoxylin & eosin (H&E) of melanomapre- and on-therapy with anti-PD-1 were evaluated independently by twodermatopathologists for the presence of tertiary lymphoid structures(TLS) at the periphery of these tumors. These are characterized byprominent lymphocytic infiltrate, sometimes with germinal centerformation and with associated high endothelial venules, as shown in FIG.10A.

RNA-seq.

Freshly isolated patient tumor samples (from patients consented toDF/HCC protocol 11-181) were snap frozen in liquid nitrogen and RNA wascollected using the Qiagen RNeasy Mini kit. RNA libraries were preparedfrom 250 ng RNA per sample using standard Illumina protocols. RNAsequencing was performed at the Broad Institute (Illumina HiSeq2000) andthe Wistar Institute (Illumina NextSeq 500). RNA samples were ribo-zerotreated and then subjected to library prep using Epicentre's ScriptSeqComplete Gold kit. Quality check was done on the Bioanalyzer using theHigh Sensitivity DNA kit and quantification was carried out using KAPAQuantification kit. Raw RNA-Seq data (BAM files) read counts weresummarized by featureCounts with parameters that only paired-ended, notchimeric and well mapped (mapping quality >/=20) reads are counted. Thennormalization was applied to eliminate bias from sequencing depths andgene lengths by edgeR, thus RPKMs (Reads Per Kilobase of transcript perMillion mapped reads).

Quantitative Real-Time PCR.

Analysis of expression levels of CCL19 and CXCL13 by quantitativereverse transcription polymerase chain reaction (qRT-PCR) was performedusing tissue samples obtained from patients with metastatic melanoma.Samples were selected from patients treated with anti-PD-1 therapy withavailable tissue pre- and on-treatment. All patients provided writtenconsent to DF/HCC protocol 11-181 (Melanoma Tissue and BloodCollection). Tissue samples were snap frozen in liquid nitrogen andprocessed to yield RNA, which was stored at −80° C. after extraction.Normal lymph node tissue was used as a positive control for expressionof CCL19 and CXCL13. Primers were designed for CXCL13 (Fwd:5′-GAGGCAGATGGAACTTGAGC-3′(SEQ ID NO: 1), Rev: 5′-CTGGGGATCTCGAATGCTA-3′(SEQ ID NO: 2)) and CCL19 (Fwd: 5′-CCAACTCTGAGTGGCACCAA-3′ (SEQ ID NO:3), Rev: 5′-TGAACACTACAGCAGGCACC-3′ (SEQ ID NO: 4)), Total RNA wasextracted via the QIAGEN RNeasy Mini Kit after being ground with theQIAGEN TissueRuptor. The extraction process was automated via the QIAGENQIAcube. RNA was stored in 1.5 mL RNAse-free EP tubes and thenquantified using the QIAGEN Qubit. cDNA was reverse-transcribed from RNAusing the Invitrogen Superscript VILO kit run on an Applied Biosystems2720 Thermo Cycler and then stored in 1.5 mL tubes in a −40° C. freezeruntil later use. For qPCR the samples were run in triplicate on a RocheLightCycler 96 using Bio-Rad's SsoAdvanced Universal SYBR Green Supermixin a total volume of 20 μL per well. 0-tubulin (Fwd:5′-CGCAGAAGAGGAGGAGGATT-3′ (SEQ ID NO: 5). Rev:5′-GAGGAAAGGGGCAGTTGAGT-3′ (SEQ ID NO: 6)) were employed to normalizethe expression of target genes. Four runs were performed. RT-PCR wasperformed in triplicate and values averaged. Effect of PD-1 blockadedepicted as log 2 fold change in CCL19 or CXCL13 expression (normalizedto β-tubulin) from on-treatment samples relative to pre-treatmentsamples. For analysis of CCL19/CXCL13 expression in sorted cellpopulations (FIG. 10H), qRT-PCR was performed according to methods knownin the art (e.g., Zhu, Z. et al. Cancer Discov. (2014), the content ofwhich is incorporated herein by reference in entirety) using thefollowing primers: and CXCL13 (Fwd: 5′-CTCTGCTTCTCATGCTGCTG-3′ (SEQ IDNO: 7). Rev: 5-TGAGGGTCCACACACACAAT-3′ (SEQ ID NO: 8)) and CCL19 (Fwd:5′-ATCCCTGGGTACATCGTGAG-3′ (SEQ ID NO: 9), Rev:5′-GCTTCATCTTGGCTGAGGTC-3′ (SEQ ID NO: 10)), using 36B4 (Fwd:5′-CAGATTGGCTACCCAACTGTT-3′ (SEQ ID NO: 11), Rev:5′-GGAAGGTGTAATCCGTCTCCAC-3′(SEQ ID NO: 12)) to normalize geneexpression.

Immunohistochemistry.

Immunohistochemistry staining was performed on 4 μm formalin-fixedparaffin-embedded sections. All procedures were done on the automatedVentana Discovery Ultra staining system. Sections were firstdeparaffinized using EZ prep solution and antigen retrieval was achievedusing Cell Conditioning solution 1. Sections were blocked with DiscoveryInhibitor (all from Ventana). Sections were incubated with primaryantibodies for 16 minutes for population markers and 12 hours for CXCL13and CCL19, then washed and incubated with OmniMap anti-Mouse oranti-Rabbit conjugated with horseradish peroxidase (HRP) (Ventana, cat#760-4310 and 760-4311) for an additional 16 minutes. Discovery Purpleor OmiMAP DAB chromogen kits (cat #760-229 or 760-159) was then appliedto generate a color reaction. Slides were then counterstained withhematoxylin II followed by bluing reagent (Ventana, cat #790-2208 andcat #760-2037). Primary antibodies used for staining were: anti-CCL19(RD Systems, cat #MAB361-100; 1:200) and anti-CXCL13 (Abcam, cat#ab112521; 1:150), CD31 (Cell Marque, cat #131M-94; 1:500): αSMA (Abcam,cat #ab5694; 1:400).

Source Data.

Data for differential CCL19 and CXCL13 gene expression analysis wereobtained from published reports (e.g., Hugo, W. et al. Cell (2016).Chen, P. L. et al. Cancer Discov. (2016), Van Allen, E. M. et al.Science (2015). Cancer Genome Atlas, N. Cell (2015), the contents ofeach of which are incorporated herein by reference in entirety). Fordata from Hugo et al, and Van Allen et al., transcriptome sequencingdata were aligned using TopHat and normalized gene expression, presentedas transcripts per million (TPM), was quantified using DESeq2 (e.g., seeKim, D. et al. Genome Biol. (2013), and Love, M. I. et al. Genome Biol.(2014), the contents of each of which are incorporated herein byreference in entirety) (Table S3). NanoString source data (Chen et al.)for PD-1 responders (n=4) and non-responders (n=8) were compared(on-PD-1/pre-PD-1) and expressed as log-2 fold change. For TCGAanalysis, raw RNA-Seq data (BAM files) of TCGA SKCM samples wasdownloaded from Genomic Data Commons and read counts were summarized(featureCounts) and normalized using edgeR, to generate RPKMs. CCL19 andCXCL13, samples were separated into two groups by kmeans clustering ofRPKM values: high group and low group, value of center of each group wasused to label high or low. The survival curves were constructedaccording to Kaplan-Meier method on these two groups (high; n=206 andlow; n=257) and survival was compared between groups using the logrank(Mantel-Cox) test (α=0.05). Four-way grouping was performed using mediancutoff to define high and low expression. Single sample GSEA (ssGSEA)was performed using immune cell signatures according to methods known inthe art (e.g., Bindea, G. et al. Immunity (2013), the content of whichis incorporated herein by reference in entirety).

Synthesis of Compound 1,5-(4-((4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)amino)-1,3,5-triazin-2-yl)-2-((tetrahydro-2H-pyran-4-yl)oxy)benzonitrile

Step 1. To a solution of 2,4-dichloro-1,3,5-triazine (9.5 g, 63 mmol) inN,N-dimethylformamide (150 mL) at 0° C. (flushed with argon balloon) wasadded a solution of 4-(4-(oxetan-3-yl)piperazin-1-yl) aniline (14.1 g,60.2 mmol) in N,N-dimethylformamide (100 mL) over 5 minutes via cannulaand stirred in an ice-bath for 1 hour. A solution of 40% Methanol/CH₂Cl₂(200 mL) was added to the reaction mixture and stirred at roomtemperature. After 1 hour, the solid formed were filtered and washedtwice with diethyl ether. Solids were collected to provide the first lotof product. To the filtrate, diethyl ether (200 mL) was added andstirred overnight at room temperature. The solids were separated byfiltration to provide a second lot of product. Both lots were combinedto provide a total yield of 20 g (91%) of4-chloro-N-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)-1,3,5-triazin-2-aminewhich was used without purification. LCMS-ESI⁺ (m/z): calculated forC₁₆H₉CIN₆O: 346.1; found: 347.1 (M+H).

Step 2. To a mixture of4-chloro-N-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)-1,3,5-triazin-2-amine(4.6 g, 13.2 mmol),2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile(3.5 g, 14.5 mmol), Pd(dppf)Cl₂CH₂Cl₂ (1.2 g, 1.6 mmol) and potassiumcarbonate (3.6 g, 26.4 mmol) under argon was added a mixture ofde-gassed solvents (1,2-dimethoxyethane (53 mL)/water (27 mL)) andsonicated until all solids went into solution (˜5 minutes). The mixturewas stirred under argon at 100° C. in a heating block for 1 hour. Aftercooling to room temperature, water (200 mL) was poured into the reactionmixture and the solids were filtered off and washed with diethyl ether(100 mL). The resulting dark brown solids were suspended in Acetonitrile(20 mL) and stirred at reflux (˜2 minutes) and then stirred at roomtemperature for 2 hours. To this suspension di-ethyl ether (20 mL) wasadded and the mixture was stirred at room temperature overnight. Solidswere taken by filtration to yield crude dark brown product. In 1 gbatches, the crude product was suspended in Dichloromethane (150 mL) ina separatory funnel. To the suspension Trifluoroacetic acid was addeduntil all solids had gone into solution. Water was added (150 mL) andmixture was shaken vigorously until black precipitates appeared. Theblack solids were filtered off. To the filtrate, a saturated aqueoussolution of NaHCO₃ was added slowly to fully neutralize the mixture. Nosolids precipitated during this process. The organic phase was driedover MgSO₄ and evaporated under reduced pressure to yield a brightyellow material (Total yield 3.1 g, 55% yield). LCMS-ESI⁺ (m/z):calculated for C₂₃H₂₂FN₇O: 431.1; found: 432.2 (M+H).

Step 3. To a solution of Tetrahydro-2H-pyran-4-ol (4.7 mL, 49 mmol) inTHF (200 mL) at 0° C., Potassium t-butoxide (3.8 g, 51 mmol) was addedand the reaction mixture was allowed to warm to room temperature. After30 minutes, solid2-fluoro-5-[4-([4-[4-(oxetan-3-yl)piperazin-1-yl]phenyl]amino)-1,3,5-triazin-2-yl]benzonitrile(10 g, 23.2 mmol) was added and stirred overnight at 60° C. The reactionmixture was then cooled to 0° C. with a water/ice bath and slowlydiluted with water (1.2 L) over 30 minutes and stirred at roomtemperature for 45 minutes. The solids formed were filtered and dried togive5-(4-((4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)amino)-1,3,5-triazin-2-yl)-2-((tetrahydro-2H-pyran-4-yl)oxy)benzonitrileas a yellow solid (10.6 g. 90% yield) LCMS-ESI⁺ (n/z): calculated forC₂₈H₃₁N₇O₃: 513.3; found: 514.5 (M+H) 1H NMR (400 MHz, DMSO-d6) δ 10.23(d, J=19.4 Hz, 1H), 8.80 (s, 1H), 8.74-8.50 (m, 2H), 7.68 (br, 2H), 7.60(d, J=9.0 Hz, 1H), 7.11 (br, 2H), 4.99 (m, 1H), 4.91-4.74 (m, 4H). 4.52(m, 1H), 3.91 (m, 2H), 3.59 (ddd, J=11.6, 8.5, 3.0 Hz, 2H), 3.80-3.30(m, 4H), 3.30-2.90 (m, 4H). 2.09 (m, 2H), 1.87-1.64 (m, 2H).

In Vitro Characterization of Compound 1.

Biochemical single point inhibition and IC₅₀ concentrations for TBK1,IKKε (IKBKE), and off-target kinases were determined at ThermoFisherScientific using SelectScreen Kinase Profiling Services (Madison, Wis.,USA) (Tables S4A and S4B-1-7). To determine cellular potency, the humancolorectal cancer carcinoma cell line HCT116 (ATCC, Manassas. Va.) wasmaintained in T175 flasks in complete RPMI medium: RPMI 1640supplemented with 10% FBS, 1×Penicillin-Streptomycin solution and 1×MEM(non-essential amino acids). HCT116 cells were grown to 90-95%confluency in T175 flasks containing complete RPMI medium andtransfected in bulk using Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) with 70 μg of ISG54-luciferase reporter plasmid (ElimBiopharmaceuticals Inc., Hayward, Calif.). The reporter plasmid containsa luciferase gene expression cassette under the transcriptionalregulation of the promoter of the human interferon stimulated gene 54(ISG54). Transfection of the cells was allowed to take place for 6hours, after which the cells were harvested by treatment with 0.25%trypsin EDTA (Corning Inc., Corning, N.Y.). Trypsinized cells were addedto 384-well poly-d-lysine treated black clear bottom tissue cultureassay plates (Greiner Bio-One GmbH, Kremsmünster, Austria) at a densityof 20,000 cells/well in 80 μL of complete RPMI medium and incubatedovernight. After 16-18 hours post-transfection, the assay plates werewashed with PBS (Corning Inc., Corning, N.Y.), followed by addition of80 μL/well of serum-free RPMI 1640 medium containing1×Penicillin-Streptomycin solution, 1×MEM and 350 nL of DMSO ortitrations of Compound 1. Compound 1 titrations were generated by1.5-fold dilution steps in two overlapping serial dilution series togenerate a 40-point compound dose range. After incubation at 37° C. for1 hour, the cells were stimulated with Poly(I:C) (InvivoGen, San Diego,Calif.) at a final concentration of 15 μg/mL in Optimem media (LifeTechnologies. Rockville, Md.). The assay plates were incubated for 5hours at 37° C., followed by addition of One-Glo luciferase fireflyreagent (Promega, Madison, Wis.) at 1:1 volume/well and luminescence wasmeasured in an EnVision Multilabel Plate Reader (PerkinElmer, SantaClara, Calif.). The EC₅₀ values were calculated from the fit of thedose-response curves to a four-parameter equation. All EC₅₀ valuesrepresent geometric mean values of a minimum of four determinations.

Interleukin-2 and Interferon Gamma Analysis.

Freshly isolated human CD4+ and CD8+ T cells were obtained from AllCells(Alameda, Calif. USA). Cells were spun down and resuspended in serumfree Xvivo 15 media (Lonza Walkersville, Inc., Chicago, Ill., USA)supplemented with 5 ng/mL IL-17 and incubated overnight at 37° C. Cellswere plated on anti-CD3 coated plates (5 μg/mL OKT3, eBioscience.Dallas, Tex. USA, overnight) with 2 μg/mL anti-CD28 (eBioscience,Dallas, Tex., USA). Cells are treated in replicate plates with adose-titration of Compound 1 for 24 hours for IL-2 and 96 hours forIFNγ. IL-2 and IFNγ in the supernatant were measured using single- ormulti-plex immunoassay (Mesoscale Discovery, Rockville, Md. USA). JurkatT cell leukemia cells (clone E61 obtained from ATCC) plated on anti-CD3coated plates were treated with a dose titration of Compound 1 for 24hours. IL-2 in the supernatant was measured as described above.

In Vivo Compound 1 Combination Treatments.

Combination studies were performed by vivoPharm (Hummelstown, Pa., USA).All procedures used in the performance of these studies were carried outin accordance with vivoPharm's Standard Operating Procedures, withparticular reference to US_SOPvP_EF0314 “General Procedures for EfficacyStudies.” CT26 colon carcinoma cells (1×10⁶ cells/mouse in 100 μL,passage 2-3) were re-suspended in serum-free DMEM and implanted in theupper right flank of 11-12 week old female Balb/c mice (Charles RiverLaboratories). Mice were randomized into four groups of 10 using amatched pair distribution method based on tumor size for CT26. Treatmentwas initiated 12 days post-inoculation with mean tumor volume at startof dosing of 125.85 mm³. Vehicle or Compound 1 (40 mg/kg) wasadministered by oral gavage daily for 26 days and isotype control or areverse chimera anti-PD-L1 cloned from literature reports (e.g., Deng,R. et al. MAbs (2016), the content of which is incorporated herein byreference in entirety) and placed into a mouse IgG1 framework (10 mg/kg)was administered every 5 days for a total of six doses. Investigatorswere not blinded to treatment groups. Mice bearing CT26 tumors withexceptional responses to combination therapy with αPD-L1 and Compound 1were re-implanted with CT26 cells (1×10⁶, lower left flank) and EMT6cells (0.5×10⁶, upper left flank) to evaluate development of immunologicmemory. MB49 and MC38 in vivo combination studies performed underidentical conditions in B6 mice. B16F10 tail vein injection studies wereperformed in accordance with methods known in the art (e.g., Zhang, J.et al. Cell Rep. (2016), the content of which is incorporated herein byreference in entirety). After tail vein injection (1×10⁵ cells in 100μL), mice were treated with Vehicle or 40 mg/kg Compound 1 daily for 13days t anti-PD-L1 (10 mg/kg) or isotype control on Days 0, 5, and 10.Mice were sacrified on Day 13 and lung metastases were quantified(small<1 mm, medium >1 mm and ≤3 mm, large >3 mm). No large lungmetastases were detected.

Statistical Methods and Data Analysis.

All graphs depict mean±s.d, unless otherwise indicated. Graphs weregenerated and statistical analysis performed using GraphPad/Prism (v7.0)and R statistical package. Pearson correlation matrix using 21 cellsurface markers for MDOTS were calculated with R across tumors anddifferent sized spheroids. Unsupervised hierarchical clustering wasperformed using GenePattern.

Example 2. Ex Vivo Profiling of PD-1 Blockade Using Organotypic TumorSpheroids

Existing patient-derived cancer models, including circulating tumorcells (CTCs), organoid cultures, and patient-derived xenografts (PDXs)can guide precision cancer therapy, but take weeks to months to generateand lack the native tumor immune microenvironment. Current approaches tostudy anti-tumor immune responses in patients are also limited by remotemeasurements in whole blood or plasma, or static assessment of biopsies.To overcome these challenges and model immune checkpoint blockade (ICB)ex vivo, a 3D microfluidic device was adapted to the short-term cultureof murine- and patient-derived organotypic tumor spheroids(MDOTS/PDOTS). Following limited collagenase digestion of fresh tumorspecimens, multicellular organotypic spheroids with autologous immunecells were isolated. MDOTS/PDOTS were analyzed by flow cytometry (FIG.5) or loaded in collagen into the central channel of the device forexposure to anti-PD-1 or anti-CTLA-4 antibodies (FIG. 1A).

Using the MC38 and B16F10 immune competent syngeneic murine tumormodels, lymphoid and myeloid immune cell composition of bulk tumor wascompared with different spheroid populations (S1 >100 μm; S2 40-100 μm;and S3<40 μm). Flow cytometric analysis revealed similar populationsacross all immune cell fractions examined (FIG. 1B, FIGS. 6A-6C. TablesS2-1 through S2-4). MDOTS (S2) from B16F10 demonstrated fewer CD45+cells than MC38 and another model, CT26 (Extended Data FIG. 2d ),although immune sub-populations were relatively consistent across MC38,B16F10, and CT26 models (FIG. 6E). Since S2 MDOTS were optimally sizedfor culture in the microfluidic device, this fraction was utilized forsubsequent studies.

A large panel of PDOTS (n=40) was immunophenotyped by flow cytometry,enriching for cancers responsive to PD-1 blockade (e.g., melanoma.Merkel cell carcinoma) (FIG. 1C, Tables 1A and 1B). A range of lymphoid(CD19+ B cells, CD4+ and CD8+ T cells) and myeloid (CD15+ granulocytic,CD14+ monocytic lineages, CD123+ dendritic cells) populations wereconsistently detected in PDOTS (FIG. 1C). Variable surface expression ofexhaustion markers (PD-1, CTLA-4, TIM-3) was detected on CD4+ and CD8+ Tcells (FIG. 6F) and PD-1 ligands (PD-L1 and PD-L2) on myeloidpopulations, including dendritic cells, myeloid-derived suppressor cells(MDSCs), and tumor-associated macrophages (TAMs) (FIG. 6G). A strongcorrelation of T-cell profiles was confirmed between PDOTS (S2) and S3fractions, including antigen-experienced (CD45RO+) (FIG. 1D) andexhausted CD4 and CD8 T cells (FIG. 1E), and overall conservation ofimmunophenotype regardless of spheroid size (FIGS. 6H-6J). These datademonstrate that PDOTS retain autologous immune cells, including keytumor-infiltrating T lymphocyte populations.

3D microfluidic culture of MDOTS and PDOTS resulted in growth andexpansion over time (FIG. 2A) as well as cytokine elaboration inconditioned medium (FIGS. 2B-2C). Tumor/immune cell intermixture inspheroids was further demonstrated by immunofluorescence microscopy(FIGS. 7A-7C). Dynamic cellular interactions were observed by liveimaging, and survival of CD45+ immune cells ex vivo in the device wasconfirmed (FIG. 7D). Thus, short-term culture and cytokine profiling ofPDOTS/MDOTS is feasible using this 3D microfluidic device.

MDOTS were exposed to ex vivo PD-1 blockade, starting with MC38allografts that respond to anti-PD-1 treatment in vivo (FIGS. 2D-2E).MC38 MDOTS were treated with anti-PD-1 antibody (or isotype control) for3 days or 6 days in the device (FIG. 2F). Dual labeling de-convolutionfluorescence microscopy using acridine orange (AO, live cells) andpropidium iodide (PI, dead cells) demonstrated >90% viability of MDOTSat the time of spheroid loading (Day 0) and over time in culture withcontrol (FIG. 2G). Treatment with anti-PD-1 resulted in dose- andtime-dependent killing of MC38 MDOTS (FIG. 2G, FIG. 2K, FIG. 7E), incontrast to cell line-derived MC38 spheroids lacking autologousimmune/stromal cells (FIG. 2H). Modest killing was evident in theintermediately sensitive CT26 model (FIG. 2I), whereas MDOTS derivedfrom the PD-1 resistant B16F10 model exhibited little cell death despiteidentical treatment (FIGS. 2J-2K). Anti-PD1 induced killing of CT26MDOTS required CD8+ T cells (FIG. 7F), which varied between tumors ofdifferent size, but not between distinct regions from a single tumor(FIG. 7G). These data demonstrate the ability to recapitulatesensitivity and resistance to PD-1 blockade ex vivo using well-definedmouse models.

Responses to PD-1 blockade in PDOTS were evaluated. To performsystematic assessment of ex vivo ICB in PDOTS, acute cytokine productionwas used as a quantitative measure of early immune activation ratherthan ex vivo killing, which was more robust in MDOTS. Day 3 cytokinerelease from PDOTS (n=28)±PD-1 blockade was analyzed, and upregulationof CCL19 and CXCL13 was observed in the majority of samples (23/28)(FIG. 3A, FIG. 3D, FIGS. 8A-8C). CCL19/CXCL13 induction was not observedwith isotype IgG control (FIG. 8D), and was comparatively minimalfollowing CTLA-4 blockade (FIG. 4B, FIG. 4E). CCL19/CXCL13 generationwas also preserved across a range of spheroid numbers (FIG. 8E) withlittle intra-assay variability (FIGS. 8F-8G). CCL19 correlated withCXCL13 across PDOTS samples, and required the microfluidic device forrobust induction (FIGS. 8H-8I). CCL19/CXCL13 upregulation was alsoevident following dual PD-1+CTLA-4 blockade (FIG. 3C. FIG. 3F), andaccompanied by induction of additional effector cytokines (e.g., IFN-γ.IL-2, and TNF-α) in select samples (FIG. 9).

In consonance with PDOTS profiling results, CCL19/CXCL13 mRNA expressionincreased in patients treated with PD-1 blockade (FIGS. 3G-3H, TablesS4A and Tables S4B-1 through S4B-7). Although fold induction ofCCL19/CXCL13 did not clearly correlate with response (FIG. 10A), higherabsolute levels of CCL19 and CXCL13, and their receptors (CCR7 andCXCR5), was evident in banked samples from melanoma patients withclinical benefit (CB) from checkpoint blockade (FIGS. 3I-3J, FIGS.10B-10C). Pretreatment CCL19/CXCL13 mRNA expression levels alone failedto correlate with responsiveness to PD-1 or CTLA-4 blockade in melanomaRNAseq datasets (FIGS. 10D-10E). NanoString data from matchedformalin-fixed and paraffin-embedded tissue or banked plasma was alsoinsufficient to detect correlation with response (FIGS. 10F-10G).

As PD-1 blockade promotes immune cell infiltration in vivo, gene setenrichment analysis (GSEA) was performed using published immunesignatures, which revealed enrichment of diverse immune cell populationsin patients with CB to checkpoint blockade (FIG. 3K). To evaluate theclinical significance of CCL19/CXCL13, Cancer Genome Atlas melanoma(SKCM) data were analyzed. Improved patient survival was evident inmelanoma specimens with higher expression of CCL19/CXCL13 (FIGS. 3L-3M;p<0.001). Evaluation of TCGA data for other cancers (e.g., bladdercancer, head and neck, and breast) revealed similar a pattern (FIG.10H). Immune GSEA using melanoma TCGA data confirmed enrichment ofdiverse immune cell gene sets in melanoma patients with high levels ofboth CCL19 and CXCL13 (FIG. 3N), consistent with their established rolesas chemoattractants.

CCL19/CXCL13 coordinate humoral and cell-mediated adaptive immunity inlymph nodes and tertiary lymphoid structures (TLS), andcancer-associated TLS have been associated with improved prognosis.Histologic review of melanoma biopsy specimens pre- and on-anti-PD1 onlyidentified rare TLS (FIG. 11A) likely due to limited sampling by corebiopsy. Expression of CCL19 in the melanoma TME has been linked tocancer-associated fibroblasts (CAFs), and CXCL13 to CD8+ exhausted Tcells. CCL19 is also strongly expressed in lymph node high endothelialvenules. It was noted that strongest induction of CCL19/CXCL13 byanti-PD1 was independent of the number of total CD45+ cells or otherimmune subpopulations in PDOTS (FIGS. 11B-11D). Therefore, CD45+ andCD45− cell populations from a sample (MGH-16) marked by robust anti-PD1induced secretion of CCL19 and CXCL13 were sorted, to determine thecellular source of CCL19 and CXCL13 in PDOTS. Indeed, stromalpopulations including CD90+CD45− cancer-associated fibroblasts,CD31+CD144+ endothelial cells, and tumor cells (CD45−) expressed CCL19in particular, while CXCL13 was also detected in CD45+ cells (FIG. 11E).Immunohistochemical staining confirmed expression of CCL19 and CXCL13 instromal elements including cancer-associated fibroblasts (αSMA) andendothelial cells (CD31) (FIG. 11F).

To better understand the relationship between anti-PD1 induced PDOTScytokine profiles and clinical benefit to ICB, unsupervised hierarchicalclustering of cytokine profiles from patients specifically treated withanti-PD-1 therapy was performed. PDOTS from patients with mixed responseor progression on anti-PD-1 therapy elaborated multiple immunesuppressive cytokines/chemokines (e.g., CCL2) in addition toCCL19/CXCL13 (FIG. 3O). Induction of significantly higher levels ofCCL20 and CX3CL1 in patients with no clinical benefit (NCB) to PD-1blockade (FIG. 12A) was noted, consistent with their lack of associationwith survival (FIG. 12B) and previous association of CX3CL1 inductionwith anti-PD-L1 resistance. Several of these immune suppressivecytokines/chemokines induced in PDOTS were also components of an innatePD-1 resistance (IPRES) gene expression signature (FIG. 12B), whichcorrelated with nonresponse but missed statistical significance (p=0.08.FIG. 12C). Serial PDOTS profiling from an individual patient revealedthat induction of multiple granulocytic and monocytic chemoattractantspredicted infiltration of these respective myeloid populations followingdisease progression on anti-PD-1 therapy in vivo (FIGS. 12D-12E). Thesedata highlight the potential of this assay to identify cytokines changesassociated with ineffective anti-tumor immune responses despiteCCL19/CXCL13 induction following ICB.

Higher levels of immune suppressive cytokines were also induced inanti-PD1-resistant B16F10 MDOTS relative to MC38 MDOTS (FIG. 12F), aswell as the intermediately resistant CT26 MDOTS model, which elaboratedparticularly high levels of CCL2 (FIG. 12G). Because of its partialsensitivity to anti-PD1, CT26 model was used to determine whether MDOTSprofiling could identify novel combination therapies that overcomeresistance. However, neutralization of CCL2 alone failed to enhance PD-1mediated killing of CT26 MDOTS (FIG. 12H-12I), suggesting the need foralternative strategies that more broadly inhibit immune suppressivesignaling within the TME and reactivate T cells.

It was noted that the homologous innate immune signaling kinases TBK1and IKKε not only promote autocrine/paracrine cytokine signaling, butalso restrain T cell activation, suggesting that a dual impact ofTBK1/IKKε inhibition on the TME could enhance anti-tumor activity ofPD-1 blockade (FIG. 4A). A novel potent/selective TBK1/IKKε inhibitorCompound 1 (Cmpd1) was synthesized (FIG. 4B, FIGS. 13A-D, Tables S5-4and S5-2), which lacks JAK inhibitory activity, in contrast to themultitargeted inhibitor momelotinib (CYT387). Cmpd1 effectively blockedimmune suppressive cytokine elaboration by CT26 cell line spheroids,without cytotoxic effects (FIG. 4C, FIG. 13E), and enhanced secretion ofIL-2 and IFN-γ from purified CD4+ and CD8+ T cells (FIGS. 4D-4E) andIL-2 from Jurkat cells (FIG. 13F). Decreased levels of CCL4, CCL3, andIL-1p with concomitant induction of cytokines involved in activatedinnate immune responses (e.g., G-CSF) were observed in CT26 MDOTStreated with Cmpd1±anti-PD-1 (FIG. 4F). Ex vivo combination treatmentenhanced killing of CT26 MDOTS (FIGS. 4G-4H), which predicted in vivoresponse with greater tumour control and longer survival than micetreated with Cmpd1 or anti-PD-L1 alone (FIGS. 41-4K). Reimplantation ofCT26 into mice with exceptional responses to combination therapy showedno growth, suggesting immunologic memory for CT26 tumors (FIG. 13F).

Combination treatment in the PD-1 sensitive MC38 MDOTS model enhanced exvivo killing and in vivo survival, although neither of these reachedsignificance compared to single-agent PD-1/PD-L1 (FIGS. 14A-14D). Theintrinsically resistant B16F10 model could not be sensitized with Cmpd1treatment (FIGS. 14E-14H), possibly due to the relative paucity ofimmune cells in B16F10 MDOTS compared to CT26 and MC38 MDOTS. Indeed,downregulation of several immune suppressive chemokines (e.g., CCL2,CCL5) in B16F10 MDOTS was observed, but without concomitant induction ofG-CSF and related innate immune response cytokines (FIGS. 14A-14B).However, enhanced response to combination treatment with Cmpd1 wasconfirmed in vivo using a fourth partially anti-PD1 sensitive syngeneicmodel (MB49 bladder carcinoma), further validating its activity.Importantly, MDOTS responses in three models (CT26, MC38, and B16F10)effectively recapitulated the in vivo response (or lack thereof) to PD-1blockade +/− TBK1/IKKε inhibition, highlighting the potential of ex vivoscreening in MDOTS to develop combination immunotherapies moregenerally.

Example 3. Transcriptomic Analysis of MDOTS/PDOTS Following Ex VivoTreatment

RNA is collected from a subset of the MDOTs within the device after thesupernatants have been collected and cDNA libraries for RNA sequencingis generated to evaluate global transcriptome changes associated withtreatment regimen on single samples per drug treatment. These types ofdata may be generated from a variety of clinical specimens. The RNAAdvance tissue RNA isolation method (Beckman Coulter) is utilized, whichis a bead wash based separation technique, which facilitates elutionvolumes amenable to sample input for RNA-Seq library preparation.Automated library preparation can be performed using as little as 10 ngof good quality RNA. The RNA Access method from Illumina is utilized,which enriches for the coding region of the genome. Libraries aresequenced on the NextSeq500 platform, as 75 bp Single End to generateapproximately 20 million reads per sample. With appropriate adaptors, 24samples are multiplexed on each NextSeq500 run. The concentration[ng/ul], yield [ng] and quality of RNA (high RIN # by bioanalyzer)derived from the MDOTS are more than sufficient to ensure good qualitylibraries for subsequent sequencing (FIG. 15).

The STAR RNA sequencing alignment tool (Spliced Transcripts Alignment toa Reference) [STAR_2.5.0a]) is utilized to align the data to the genome(RefSeq gene annotations). DeSeq2 is utilized to perform differentialexpression analysis (±2-fold, P value<0.05). Overlaps betweendifferentially expressed genes and the annotated Immunological gene setsin the Molecular Signature Database or MSigDB (FDR q-value<0.05) arecomputed. CIBERSORT (Newman A M et al. Robust enumeration of cellsubsets from tissue expression profiles. Nat Methods. 2015 May;12(5):453-7.) is utilized to provide an estimation of the abundances ofmember cell types in a mixed cell population, including more than 20immune subsets, using the transcriptomic data generated.

The additional information garnered from whole transcriptome analysisprovides a deeper understanding of the biological response to PD-1immune blockage on its own and in combination with other immune blockadecompounds or a MEK inhibitor. This knowledge complements secretionprofiling to identify novel mechanisms of both response and resistance.Moreover, computational methods to evaluate the “immunone” includingGSEA and CIBERSORT, permit inference of changes in immune cellpopulations using established gene signatures.

Example 4. RNA-Sequencing of PDOTS/MDOTS for Biomarker Discovery andNovel Target Identification

Bulk RNA-seq was applied to the MDOTS/PDOTS platform as aproof-of-concept to identify candidate biomarkers and potential noveltargets for combination therapies in response to inhibition of PD-1.

The patient- and murine-derived organotypic tumor spheroids(PDOTS/MDOTS) platform was utilized to identify novel biomarkers andpotential therapeutic targets using RNA-sequencing (RNA-seq).Reproducible induction of cytokines and chemokines (e.g. SOCS3, Pde4b,Pde4d, Adcy5) in response to ex vivo PD-1 blockade using MDOTS and PDOTSwas observed (see FIG. 16). Additionally, dual-labeling fluorescencemicroscopy was used to demonstrate immune-mediated tumor killing. Thismethod for imaging and quantifying immune-mediated tumor killing incombinatorial drug testing represented a novel use of availablefluorescent reagents. Evaluation of combinations of different targetedtherapies revealed that some therapeutic agents appear to sensitizePDOTS/MDOTS to PD-1 blockade resulting in enhanced immune responses,manifested by alterations in cytokine elaboration, and enhanced tumorkilling. In particular, combined TBK1-PD-1 therapy was found to be anovel therapeutic combination to enhance PD-1 response.

Tables with Supportive Data for Example 2:

TABLE S2-1 Tumor Tumor Tumor Tumor Tumor 1.000 2.000 4.000 5.000 6.000Tumor 1 1.000 0.955 0.890 0.752 0.240 Tumor 2 0.955 1.000 0.835 0.7120.223 Tumor 4 0.890 0.835 1.000 0.907 0.495 Tumor 5 0.752 0.712 0.9071.000 0.758 Tumor 6 0.240 0.223 0.495 0.758 1.000 S1 1 0.852 0.900 0.6190.555 0.197 S1 7 0.975 0.963 0.852 0.699 0.129 S1 3 0.948 0.967 0.7560.611 0.087 S1 4 0.838 0.821 0.915 0.911 0.676 S1 5 0.558 0.549 0.7080.802 0.860 S1 6 0.461 0.470 0.554 0.691 0.834 S2 1 0.749 0.788 0.6030.635 0.455 S2 2 0.948 0.936 0.865 0.816 0.367 S2 3 0.881 0.883 0.6960.634 0.230 S2 4 0.680 0.651 0.836 0.925 0.825 S2 5 0.308 0.310 0.5400.713 0.925 S2 6 0.183 0.226 0.383 0.590 0.884 S3 1 0.740 0.776 0.5690.636 0.459 S3 2 0.827 0.856 0.664 0.688 0.407 S3 3 0.790 0.817 0.5480.510 0.165 S3 4 0.685 0.675 0.792 0.839 0.768 S3 5 0.300 0.278 0.5180.567 0.793 S3 6 0.226 0.246 0.430 0.499 0.766

TABLE S2-2 S1 S1 S1 S1 S1 S1 1.000 2.000 3.000 4.000 5.000 6.000 Tumor 10.852 0.975 0.948 0.838 0.558 0.461 Tumor 2 0.900 0.963 0.967 0.8210.549 0.470 Tumor 4 0.619 0.852 0.756 0.915 0.708 0.554 Tumor 5 0.5550.699 0.611 0.911 0.802 0.691 Tumor 6 0.197 0.129 0.087 0.676 0.8600.834 S1 1 1.000 0.854 0.932 0.745 0.546 0.553 S1 2 0.854 1.000 0.9630.776 0.465 0.371 S1 3 0.932 0.963 1.000 0.737 0.447 0.399 S1 4 0.7450.776 0.737 1.000 0.897 0.803 S1 5 0.546 0.465 0.447 0.897 1.000 0.967S1 6 0.553 0.371 0.399 0.803 0.967 1.000 S2 1 0.908 0.692 0.778 0.8000.677 0.672 S2 2 0.862 0.937 0.906 0.863 0.605 0.514 S2 3 0.915 0.8490.927 0.742 0.493 0.460 S2 4 0.590 0.598 0.547 0.957 0.928 0.835 S2 50.290 0.191 0.154 0.749 0.890 0.825 S2 6 0.248 0.081 0.057 0.640 0.8220.790 S3 1 0.908 0.685 0.776 0.767 0.654 0.666 S3 2 0.937 0.808 0.8440.808 0.649 0.634 S3 3 0.921 0.764 0.888 0.642 0.428 0.437 S3 4 0.6630.605 0.580 0.959 0.958 0.889 S3 5 0.283 0.178 0.166 0.720 0.911 0.867S3 6 0.261 0.112 0.101 0.665 0.845 0.794

TABLE S2-3 S1 S2 S2 S2 S2 S2 1.000 2.000 3.000 4.000 5.000 6.000 Tumor 10.749 0.948 0.881 0.680 0.308 0.183 Tumor 7 0.788 0.936 0.883 0.6510.310 0.226 Tumor 4 0.603 0.865 0.696 0.836 0.540 0.383 Tumor 5 0.6350.816 0.634 0.925 0.713 0.590 Tumor 6 0.455 0.367 0.230 0.825 0.9250.884 S1 1 0.908 0.862 0.915 0.590 0.290 0.248 S1 2 0.692 0.937 0.8490.598 0.191 0.081 S1 3 0.778 0.906 0.927 0.547 0.154 0.057 S1 4 0.8000.863 0.742 0.957 0.749 0.640 S1 5 0.677 0.605 0.493 0.928 0.890 0.822S1 6 0.672 0.514 0.460 0.835 0.825 0.790 S2 1 1.000 0.833 0.898 0.7350.567 0.527 S2 2 0.833 1.000 0.901 0.751 0.425 0.323 S2 3 0.898 0.9011.000 0.616 0.290 0.184 S2 4 0.735 0.751 0.616 1.000 0.872 0.767 S2 50.567 0.425 0.290 0.872 1.000 0.970 S2 6 0.527 0.323 0.184 0.767 0.9701.000 S3 1 0.984 0.835 0.901 0.707 0.529 0.493 S3 2 0.945 0.920 0.8900.717 0.481 0.443 S3 3 0.893 0.816 0.976 0.517 0.217 0.139 S3 4 0.7790.741 0.625 0.970 0.862 0.782 S3 5 0.485 0.327 0.243 0.792 0.894 0.836S3 6 0.510 0.298 0.201 0.747 0.929 0.920

TABLE S2-4 S3 S3 S3 S3 S3 S3 1.000 2.000 3.000 4.000 5.000 6.000 Tumor 10.740 0.827 0.790 0.685 0.300 0.226 Tumor 2 0.776 0.856 0.817 0.6750.278 0.246 Tumor 4 0.569 0.664 0.548 0.792 0.518 0.430 Tumor 5 0.6360.688 0.510 0.839 0.567 0.499 Tumor 6 0.459 0.407 0.165 0.768 0.7930.766 S1 1 0.908 0.937 0.921 0.663 0.283 0.261 S1 2 0.685 0.808 0.7640.605 0.178 0.112 S1 3 0.776 0.844 0.888 0.580 0.166 0.101 S1 4 0.7670.808 0.642 0.959 0.720 0.665 S1 5 0.654 0.649 0.428 0.958 0.911 0.845S1 6 0.666 0.634 0.437 0.889 0.867 0.794 S2 1 0.984 0.945 0.893 0.7790.485 0.510 S2 2 0.835 0.920 0.816 0.741 0.327 0.298 S2 3 0.901 0.8900.976 0.625 0.243 0.201 S2 4 0.707 0.717 0.517 0.970 0.792 0.747 S2 50.529 0.481 0.217 0.862 0.894 0.929 S2 6 0.493 0.443 0.139 0.782 0.8360.920 S3 1 1.000 0.960 0.913 0.746 0.426 0.440 S3 7 0.960 1.000 0.8740.763 0.387 0.394 S3 3 0.913 0.874 1.000 0.550 0.181 0.146 S3 4 0.7460.763 0.550 1.000 0.849 0.812 S3 5 0.426 0.387 0.181 0.849 1.000 0.955S3 6 0.440 0.394 0.146 0.812 0.955 1.000

Table S3 Transcripts Per Million Normalized Counts CCL19 CXCL13 IDResponse CCL19 CXCL13 Pt ID Response (ENSG00000172724) (ENSG00000156234)Pt13 R 34.698109 2519.38443 Pt13 R 1 .168351014 1.450293862 Pt15 R368.803957 958.479364 Pt15 R 7.162930953 0.318252625 Pt19 R 1958.97151620.707828 Pt19 R 13.49446269 0.073098545 Pt2 R 290.527532 1198.6162Pt12 R 7.401911739 0.522072463 Pt27A R 209.760872 1416.41918 Pt27A R7.057272238 0.814700951 Pt27B R 127.405123 460.732479 Pt27B R4.111967591 0.254217624 Pt28 R 1952.44983 2324.597 Pt28 R 40.566168380.825706707 Pt35 R 95.3399393 539.624057 Pt35 R 3.445558222 0.333403426Pt37 R 97.0628571 349.174174 Pt37 R 2.033195626 0.125043823 Pt38 R9077.47447 44808.3318 Pt38 R 292.4756557 24.68183797 Pt4 R 158.462804383.601214 Pt4 R 2.601765411 0.107674982 Pt5 R 0.8957807 94.056973 Pt5 R0.038933398 0.069888488 Pt6 R 1.44330175 90.2063594 Pt6 R 0.0207244710.022144073 Pt8 R 617.842921 243.736198 Pt8 R 12.63014518 0.085181314Pt9 R 1131.85131 641.694215 Pt9 R 35.53789234 0.344448618 Pt1 NR2340.48615 479.579124 Pt1 NR 99.54999655 0.348730135 Pt10 NR 2.700896322584.08255 Pt10 NR 0.098714409 1.614630139 Pt12 NR 292.02708 93.1555918Pt12 NR 8.617132617 0.046994022 Pt16 NR 14.8144533 421.153742 Pt14 NR9.131829845 0.112841472 Pt14 NR 221.968265 160.438202 Pt16 NR0.360470362 0.17519376 Pt20 NR 3001.54512 6437.79678 Pt20 NR 49.362562711.810023107 Pt22 NR 1426.52919 960.941811 Pt22 NR 21.092942520.242911252 Pt23 NR 15.1942229 2322.92855 Pt23 NR 0.2290442850.598647058 Pt25 NR 944.629262 879.267764 Pt25 NR 11.4526496 0.182246738Pt29 NR 420.240506 1498.70041 Pt29 NR 11.97953537 0.73038345 Pt31 NR39.9689397 296.01996 Pt31 NR 0.909356479 0.115140091 Pt32 NR 4.86573555259.830278 Pt32 NR 0.18150561 0.165701082 Pt7 NR 217.069727 429.20605Pt7 NR 4.459248487 0.150737839 15 responders 13 non-responders

TABLE S4A Melanoma Patient Samples-Patient Treatment Details SamplesTreatment Clinical Benefit/Response 148-S9 pre-PD-1-combo (on trial) NCB(mixed response → 148-S10 post-PD-1-combo (on trial) (209 days)progressive disease) 208-S11 pre-CTLA-4; pre-PD-1 NCB (progression)208-S12 on-CTLA-4 (42 days); pre-PD-1 208-S13 post-CTLA-4 (182 days);pre-PD-1 208-S14 post-CTLA-4 (245 days); on-PD-1 (63 days) 27-S1post-BRAFi (479 days); pre-PD-1 NCB (progression) 27-S2 post-BRAFi (960days); on-PD-1 (31 days) 39-S15 post-BRAFi (393 days); post-IL2 (388days); post-CTLA-4 NCB (mixed response → (153 days); pre-BRAFi/MEKi;pre-PD-1 progressive disease 39-S16 post-BRAFi (540 days); post-IL2 (535days); post-CTLA-4 (300 days); post-BRAFi/MEKi (54 days); on-PD-1 (21days) 39-S17 post-BRAFi (607 days); post-IL2 (602 days); post-CTLA-4(367 days); post-BRAFi/MEKi (121 days); on-PD-1 (88 days) 42-S3post-CTLA-4 (176 days); post-BRAPi/MEKi (64 days); pre- NCB(progression) PD-1 42-S4 post-CTLA-4 (214 days); post-BRAFi/MEKi (102days); on-PD-1 (38 days) 42-S5 post-CTLA-4 (250 days); post-BRAFi/MEKi(138 days); post- PD-1 (74 days) 62-S6 pre-CTLA-4; pre-PD-1 NCB (stabledisease → disease 62-S7 post-CTLA-4 (235 days); on-PD-1 (3 days)progression) 62-S8 post-CTLA-4 (277 days); on-PD-1 (45 days) 272_S1pre-CTLA-4 CB (near complete response, 272-S2 post-CTLA-4 (47 days);pre-PD-1 PD-1 stopped due to irAE, 272-S3 post-CTLA-4 (437 days);post-PD-1 (346 days) eventual progression off therapy) 422-S1 pre-PD-1,pre-CTLA-4 CB (excellent response after ipi- 422-S7 on-PD-1, on-CTLA-4nivo → nivo) 51-S1 pie-PD-L1 CB (stable disease with a single 51-S2Post-PD-L1 (224 days) escape lesion) 98-S1 post-PD-1 (83 days);pre-CTLA-4 CB (mixed response → 98-S2 post-PD-1 (109 days); post-CTLA-4(25 days) response) 98-S3 post-PD-1 (182 days); post-CTLA-4 (98 days)

TABLE S4B-1 Samples 148-S9 148-S10 208-S11 208-S12 pre-PD- post-PD-on-CTLA-4 1-combo 1-combo (on pre-CTLA-4; (42 days); Treatment (ontrial) trial) (209 days) pre-PD-1 pre-PD-1 CCL19 0.15687084 0.419133441.98815132 4.59582353 CXCL13 0.26803097 2.38711763 0.09436038 24.7654719CCL2 0.1 0.1 0.1 0.1 CCL7 0.1 0.352523 0.13934883 0.1 CCL8 19.552641929.918033 0.90971542 3.97934158 CCL13 24.0118925 82.8961538 3.069916729.11382191 IL10 2.70808608 3.17659032 0.41855817 1.41409955 VEGFA3.53392266 4.83525269 1.29428459 0.86705549 VEGFC 6.12754266 8.32347830.81574911 2.3208504 CCR7 0.43889039 0.90613397 0.42139498 3.5517162CXCR5 0.06942884 0.13912696 0.18331834 0.39116295 AXL 32.503153560.0635857 2.91859483 8.55069013 GZMA 2.51550488 9.76148303 3.1628008553.720088 GZMB 3.13000293 3.09735564 0.7346123 33.0483114 IFNGR151.4518655 56.8050947 54.1265708 61.4638002 TNF 0.1 0.1 0.1 0.1 1L20.13457249 0.17977794 0.1 0.1 IFNG 0.26670627 0.23753197 0.1 3.00525274208-S13 208-S14 27-S1 27-S2 post-CTLA-4 post-BRAFi post-CTLA-4 (245days); post-BRAFi (960 days); (182 days); on-PD-1 (479 days); on-PD-1Treatment pre-PD-1 (63 days) pre-PD-1 (31 days) CCL19 3.455135268.77085298 0.1 0.93715502 CXCL13 2.17496489 11.6557472 1.274183460.6404927 CCL2 0.1 0.1 0.1 0.1 CCL7 0.1 0.32786454 0.1 0.1 CCL85.89116018 8.66865469 0.36852681 3.80785605 CCL13 6.00656079 10.36343720.36047157 0.30199656 IL10 0.68911285 1.88752859 0.09419912 0.23675495VEGFA 1.65186515 1.23934008 1.5851421 2.89436628 VEGFC 1.850419971.47147983 1.248408 2.33787985 CCR7 0.97129671 4.51119789 0.113805070.42904724 CXCR5 0.20120982 0.60384444 0.1 0.04147717 AXL 6.305190424.63247743 2.56483473 3.61622621 GZMA 30.132477 28.5755234 1.5375052913.8827918 GZMB 4.43469902 8.64210075 3.47191043 10.5267372 IFNGR158.8209997 82.2106075 43.6363806 28.2519413 TNF 0.1 0.1 0.1 0.1 1L2 0.10.16720274 0.1 0.1 IFNG 0.72140516 2.09871115 0.38036581 1.06221195 0.1Below quantification

Table S4B-2 Samples 39-S15 39-S16 39-S17 42-S3 post-BRAFi (607post-BRAFi post-BRAFi (540 days); days); post-IL2 (393 days); post-post-IL2 (535 days); (602 days); post- IL2 (388 days); post-CTLA-4post-CTLA-4 CTLA-4 (176 post-CTLA-4 (300 days); (367 days); days); post-(153 days); post-BRAFi/MEKi post-BRAFi/ BRAFi/MEKi pre-BRAFi/ (54 days);MEKi (121 days); (64 days); Treatment MEKi; pre-PD-1 on-PD-1 (21 days)on-PD-1(88 days) pre-PD-1 CCL19 0.1 7.17238179 0.57691197 1.23267021CXCL13 1.62907579 2.78518137 13.80004 0.23401686 CCL2 0.1 0.1 0.1 0.1CCL7 0.2830323 0.41130796 0.48522669 0.1 CCL8 3.97261692 2.506144582.32299537 2.93296153 CCL13 2.57546133 5.90954174 1.08446841 3.6412366IL10 1.9128064 1.50997511 6.1537277 0.34601269 VEGFA 2.445422961.59918098 1.08303658 0.29859241 VEGFC 0.99412394 1.76571995 1.009962940.60692541 CCR7 0.21397451 0.8706652 0.88040393 0.52253673 CXCR50.0372339 0.14429073 0.17022212 0.0909271 AXL 3.15217802 5.99379035.59225163 2.69996776 GZMA 5.26766769 14.8122602 18.7958475 3.45129418GZMB 3.6058497 22.4059016 21.3166185 3.64372548 IFNGR1 81.0920132110.98998 60.1500121 79.9433286 TNF 0.1 0.1 0.1 0.1 IL2 0.14433941 0.10.82484505 0.1 IFNG 0.66748061 0.46190268 3.16050183 0.11643014 42-S442-S5 62-S6 62-S7 post-CTLA-4 post-CTLA-4 (214 days); post- (250 days);post- BRAFi/MEKi BRAFi/ MEKi post-CTLA-4 (102 days); on- (138 days);post- pre-CTLA-4; (235 days); Treatment PD-1 (38 days) PD-1 (74 days)pre-PD-1 on-PD-1(3 days) CCL19 5.18691585 6.33513418 0.1 0.19283266CXCL13 21.2470659 3.96889863 0.1 0.98842702 CCL2 0.1 0.1 0.1 0.1 CCL70.83895949 1.24327654 0.1 0.1 CCL8 34.1765182 59.2506107 7.981166734.1293583 CCL13 67.6625745 58.3524709 5.93524222 8.85494803 IL105.87990461 2.48959659 0.16767663 0.64954159 VEGFA 2.57328173 0.920744090.72348459 1.68157005 VEGFC 3.9945039 0.90110433 1.56860848 1.77229407CCR7 0.9133338 0.53710072 0.20257569 0.09809159 CXCR5 0.441472410.0934614 0.1 0.1 AXL 10.0967011 3.89711811 4.64898024 4.8257931 GZMA118.36424 42.5698517 8.66651831 5.88984997 GZMB 109.390225 31.83489991.03001359 2.13752208 IFNGR1 95.0516258 75.0677163 0.1 35.6153856 TNF0.1 0.1 0.1 0.1 IL2 0.1 0.36230859 0.1 0.1 IFNG 7.80107938 4.308308720.56421667 0.32784729 0.1 Below quantification

TABLE S4B-3 Sam- 272- 272- 272- 422- 422- ples 62-S8 S1 S2 S3 S1 S251-S1 51-S2 98-S1 98-S2 98-S3 post- post- post- post- CTLA-4 post-CTLA-4 post- PD-1 PD-1 (277 days); CTLA-4 (437 days); pre- on- post-PD-1 (109 days); (182 days); on- (47 days); post- PD-1, PD-1, pre- PD-(83 days); post- post- Treat- PD-1 pre- pre- PD-1 pre- on- PD- L1 pre-CTLA-4 CTLA-4 ment (45 days) CTLA-4 PD-1 (346 days) CTLA-4 CTLA-4 L1(224 days) CTLA-4 (25 days) (98 days) CCL- 0.18082453 30.802674781.8439915 30.3310126 49.1698251 569.522382 31.365808 32.41527980.17967977 0.53928875 47.9201706 19 CXC- 0.20597232 143.121621197.444029 42.1785257 88.565516 236.913118 16.4200002 79.66342461.33034424 1.36021174 68.8004261 L13 CC- 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.10.1 0.1 0.1 L2 CC- 0.1 2.26838847 2.20958272 4.55339377 27.1500865 0.10.85296606 0.06224591 0.1511243 0.25919012 0.48076933 L7 CC- 0.0992875338.0092355 21.2212318 12.1190886 142.838439 3.00565688 6.9605570712.0283136 1.28256651 2.15740095 18.2040065 L8 CCL- 1.16540776 7.738094810.0937847 4.97652335 13.0430495 2.22349876 24.9771521 3.040719840.79614548 0.49652815 23.4856381 13 IL10 0.22840994 10.917529812.1675798 3.6530185 2.56684581 2.03367129 2.3790281 1.433408190.26479124 0.55145316 2.32656264 VEG- 1.21549229 1.59027526 1.899919794.36289443 1.41618926 0.51813138 0.97385307 0.71259811 2.693061426.84424133 0.76154607 FA VEG- 0.94967388 1.66183682 2.86889762 1.7314751.96825775 2.46115994 6.2059211 0.70448349 0.8551934 1.011533975.75394032 FC CC- 0.3219412 8.27974664 12.7854505 4.44863917 5.8463197695.5634831 5.96945609 8.15050896 0.13710131 0.05878489 4.26465798 R7CXC- 0.52019769 1.09942208 2.21362828 0.79868685 0.37596741 48.11809120.30457195 1.75237544 0.01988094 0.01704869 0.92762141 R5 AXL 2.452246756.6522478 74.8405834 13.3027932 10.9127259 19.1432302 2.5444273929.894754 0.48020963 0.52945552 144.038705 GZ- 3.58999916 80.8064715125.257307 46.3160997 41.8012 37.8345867 10.1174052 40.96342291.79255175 1 .87878039 27.1754305 MA GZ- 7.61677215 17.166425616.4009527 3.82328583 10.1578792 4.1352438 8.29444931 14.84781812.44928242 3.15592022 18.753749 MB IFN- 11.8733792 57.0563904 61.182691137.3127656 44.6747933 85.6737313 184.213974 47.254564 22.869931432.4953981 124.279512 GR1 TNF 0.1 1.26258841 1.58476345 0.694073511.36758178 2.61714127 0.33169142 0.45183519 0.11821037 0.040548030.73206497 IL2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 IFNG0.20495433 10.2969456 11.6529517 2.83209437 2.88850742 1.746624820.12315714 4.90717196 0.0509142 0.04366094 10.0962879 0.1 Belowquantification

TABLE S4B-4 FC 148 208a 208b 208c 27 39a 39b 42a 42b CCL19 2.67 2.311.74 4.41 9.37 71.72 5.77 4.21 5.14 CXCL13 8.91 262.46 23.05 123.52 0.501.71 8.47 90.79 16.96 CCL2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00CCL7 3.53 0.72 0.72 2.35 1.00 1.45 1.71 8.39 12.43 CCL8 1.53 4.37 6.489.53 10.33 0.63 0.58 11.65 20.20 CCL13 3.45 2.97 1.96 3.38 0.84 2.290.42 18.58 16.03 IL10 1.17 3.38 1.65 4.51 2.51 0.79 3.22 16.99 7.20VEGFA 1.37 0.67 1.28 0.96 1.83 0.65 0.44 8.62 3.08 VEGFC 1.36 2.85 2.271.80 1.87 1.78 1.02 6.58 1.48 CCR7 2.06 8.43 2.30 10.71 3.77 4.07 4.111.75 1.03 CXCR5 2.00 2.13 1.10 3.29 0.41 3.88 4.57 4.86 1.03 AXL 1.852.93 2.16 1.59 1.41 1.90 1.77 3.74 1.44 GZMA 3.88 16.98 9.53 9.03 9.032.81 3.57 34.30 12.33 GZMB 0.99 44.99 6.04 11.76 3.03 6.21 5.91 30.028.74 IFNGR1 1.10 1.14 1.09 1.52 0.65 1.37 0.74 1.19 0.94 TNF 1.00 1.001.00 1.00 1.00 1.00 1.00 1.00 1.00 IL2 1.34 1.00 1.00 1.67 1.00 0.695.71 1.00 3.62 IFNG 0.89 30.05 7.21 20.99 2.79 0.69 4.73 67.00 37.00

TABLE S4B-5 FC 62a 62b 272a 272b 422 51 98a 98b CCL- 1.93 1.81 2.66 0.9811.58 1.03 3.00 266.70 19 CXC- 9.88 2.06 1.38 0.29 2.68 4.85 1.02 51.72L13 CCL- 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2 CCL- 1.00 1.00 0.972.01 0.00 0.07 1.72 3.18 7 CCL- 0.52 0.01 0.56 0.32 0.02 1.73 1.68 14.198 CCL- 1.49 0.20 1.30 0.64 0.17 0.12 0.62 29.50 13 IL10 3.87 1.36 1.110.33 0.79 0.60 2.08 8.79 VEG- 2.32 1.68 1.19 2.74 0.37 0.73 2.54 0.28 FAVEG- 1.13 0.61 1.73 1.04 1.25 0.11 1.18 6.73 FC CCR- 0.48 1.59 1.54 0.5416.35 1.37 0.43 31.11 7 CXC- 1.00 5.20 2.01 0.73 127.98 5.75 0.86 46.66R5 AXL 1.04 0.53 1.32 0.23 1.75 11.75 1.10 299.95 GZ- 0.68 0.41 1.550.57 0.91 4.05 1.05 15.16 MA GZ- 2.08 7.39 0.96 0.22 0.41 1.79 1.29 7.66MB IFN- 356.15 118.73 1.07 0.65 1.92 0.26 1.42 5.43 GR1 TNF 1.00 1.001.26 0.55 1.91 1.36 0.34 6.19 IL2 1.00 1.00 1.00 1.00 1.00 1.00 1.001.00 IF- 0.58 0.36 1.13 0.28 0.60 39.84 0.86 198.30 NG

TABLE S4B-6 L2FC 148 208a 208b 208c 27 39a 39b 42a 42b CCL19 1.42 1.210.80 2.14 3.23 6.16 2.53 2.07 2.36 CXCL13 3.15 8.04 4.53 6.95 −0.99 0.773.08 6.50 4.08 CCL2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 CCL71.82 −0.48 −0.48 1.23 0.00 0.54 0.78 3.07 3.64 CCL8 0.61 2.13 2.70 3.253.37 −0.66 −0.77 3.54 4.34 CCL13 1.79 1.57 0.97 1.76 −0.26 1.20 −1.254.22 4.00 IL10 0.23 1.76 0.72 2.17 1.33 −0.34 1.69 4.09 2.85 VEGFA 0.45−0.58 0.35 −0.06 0.87 −0.61 −1.18 3.11 1.62 VEGFC 0.44 1.51 1.18 0.850.91 0.83 0.02 2.72 0.57 CCR7 1.05 3.08 1.20 3.42 1.91 2.02 2.04 0.810.04 CXCR5 1.00 1.09 0.13 1.72 −1.27 1.95 2.19 2.28 0.04 AXL 0.89 1.551.11 0.67 0.50 0.93 0.83 1.90 0.53 GZMA 1.96 4.09 3.25 3.18 3.17 1.491.84 5.10 3.62 GZMB −0.02 5.49 2.59 3.56 1.60 2.64 2.56 4.91 3.13 IFNGR10.14 0.18 0.12 0.60 −0.63 0.45 −0.43 0.25 −0.09 TNF 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 IL2 0.42 0.00 0.00 0.74 0.00 −0.53 2.51 0.001.86 IFNG −0.17 4.91 2.85 4.39 1.48 −0.53 2.24 6.07 5.21

TABLE S4B-7 L2FC 62a 62b 272a 272b 422 51 98a 98b CCL19 0.95 0.85 1.41−0.02 3.53 0.05 1.59 8.06 CXCL13 3.31 1.04 0.46 −1.76 1.42 2.28 0.035.69 CCL2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 CCL7 0.00 0.00 −0.041.01 −8.08 −3.78 0.78 1.67 CCL8 −0.95 −6.33 −0.84 −1.65 −5.57 0.79 0.753.83 CCL13 0.58 −2.35 0.38 −0.64 −2.55 −3.04 −0.68 4.88 IL10 1.95 0.450.16 −1.58 −0.34 −0.73 1.06 3.14 VEGFA 1.22 0.75 0.26 1.46 −1.45 −0.451.35 −1.82 VEGFC 0.18 −0.72 0.79 0.06 0.32 −3.14 0.24 2.75 CCR7 −1.050.67 0.63 −0.90 4.03 0.45 −1.22 4.96 CXCR5 0.00 2.38 1.01 −0.46 7.002.52 −0.22 5.54 AXL 0.05 −0.92 0.40 −2.09 0.81 3.55 0.14 8.23 GZMA −0.56−1.27 0.63 −0.80 −0.14 2.02 0.07 3.92 GZMB 1.05 2.89 −0.07 −2.17 −1.300.84 0.37 2.94 IFNGR1 8.48 6.89 0.10 −0.61 0.94 −1.96 0.51 2.44 TNF 0.000.00 0.33 −0.86 0.94 0.45 −1.54 2.63 IL2 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 IFNG −0.78 −1.46 0.18 −1.86 −0.73 5.32 −0.22 7.63

TABLE S5-1 Project SSBK10488_30712; provides data for Cmpd1 (1000 μM). %Inhibition % Inhibition % Inhibition ATP Kinase mutant Technology 1 2Avg Dup Difference Donor Interference Acceptor Interference Z′ KinasePart# / Lot# Dev Reaction Interference Km TBK1 ZLYTE 91 99 95 8 PassPass 0.83 PV3504/857011 Pass app CDC7/DBF4 LanthaScreen 100 97 98 3 PassPass 0.84 PV6274/1576890 Binding MAPK15 (ERK7) LanthaScreen 100 100 1000 Pass Pass 0.93 PV6181/1570972 Binding Km IKBKE (IKK ZLYTE 106 103 1053 Pass Pass 0.8 PV4875/853377 Pass app epsilon) Km CSF1R (FMS) ZLYTE 9196 93 6 Pass Pass 0.79 PV3249/662393 Pass app STK17A (DRAK1)LanthaScreen 101 101 101 0 Pass Pass 0.9 PV3783/902537 Binding TGFBR2LanthaScreen 92 95 93 3 Pass Pass 0.79 PV6122/862447 Binding PLK4LanthaScreen 96 95 96 1 Pass Pass 0.96 PV6394/1577044 Binding Km LRRK2Adapta 99 100 100 1 Pass Pass 0.76 PV4873/1106714 app Km STK22D (TSSK1)ZLYTE 99 95 97 3 Pass Pass 0.88 PV3505/1211826 Pass app Km PRKD2 (PKD2)ZLYTE 99 97 98 2 Pass Pass 0.8 PV3758/34015 Pass app Km MKNK1 (MNK1)ZLYTE 99 100 100 1 Pass Pass 0.77 PV6023/1405297 Pass app Km PRKD1 (PKCmu) ZLYTE 100 95 98 5 Pass Pass 0.86 PV3791/34226 Pass app Km MARK4ZLYTE 96 93 95 3 Pass Pass 0.8 PV3851/304213 Pass app Km MARK3 ZLYTE 9894 96 4 Pass Pass 0.73 PV4819/830446 Pass app Km PAK4 ZLYTE 91 90 90 1Pass Pass 0.73 PV4212/35324 Pass app MLCK (MLCK2) LanthaScreen 92 93 930 Pass Pass 0.56 PV3835/34028 Binding ULK3 LanthaScreen 101 104 102 3Pass Pass 0.82 PV6436/1579414 Binding Km IRAK1 Adapta 94 96 95 1 PassPass 0.91 PV4403/880118 app Km BRSK1(SAD1) ZLYTE 92 89 90 4 Pass Pass0.91 PV4333/36097 Pass app SIK3 LanthaScreen 98 95 96 2 Pass Pass 0.73PV6403/1577057 Binding Km PRKCN (PKD3) ZLYTE 98 96 97 2 Pass Pass 0.74PV3692/1252690 Pass app STK16 (PKL12) LanthaScreen 95 93 94 2 Pass Pass0.76 PV4311/36847 Binding ULK2 LanthaScreen 86 90 88 4 Pass Pass 0.9PV6433/1579413 Binding Km LRRK2 G2019S y Adapta 99 100 100 1 Pass Pass0.88 PV4881/933637 app Km LRRK2 G2019S FL y Adapta 100 99 99 1 Pass Pass0.81 A15200PT/50078 app Km LRRK2 I2020T y Adapta 100 98 99 2 Pass Pass0.84 PV5854/902533 app Km LRRK2 FL Adapta 98 98 98 0 Pass Pass 0.88A15197PT/1147693 app Km LRRK2 R1441C y Adapta 99 95 97 4 Pass Pass 0.8PV5858/902531 app KIT D816H y LanthaScreen 90 94 92 4 Pass Pass 0.82PV6196/1570980 Binding ULK1 LanthaScreen 91 92 92 1 Pass Pass 0.8PV6430/1579415 Binding ACVR1 (ALK2) y LanthaScreen 88 93 91 5 Pass Pass0.88 PV6232/1578464 R206H Binding KIT A829P y LanthaScreen 91 90 90 0Pass Pass 0.89 PV6193/1570981 Binding Km AURKA (Aurora ZLYTE 91 86 89 5Pass Pass 0.84 PV3612/32155 Pass app A) EIF2AK2 (PKR) LanthaScreen 90 8989 1 Pass Pass 0.8 PV4821/374655 Binding STK17B (DRAK2) LanthaScreen 9484 89 10 Pass Pass 0.6 PV6328/1575526 Binding CLK4 LanthaScreen 87 89 882 Pass Pass 0.81 PV3839/827665 Binding Km SNFILK2 ZLYTE 88 88 88 0 PassPass 0.66 PV4792/719848 Pass app MKNK2 (MNK2) LanthaScreen 86 88 87 2Pass Pass 0.86 PV5607/811381 Binding Km MAP4K4 (HGK) ZLYTE 86 86 86 0Pass Pass 0.74 PV3687/792773 Pass app Km MARK1 (MARK) ZLYTE 85 87 86 3Pass Pass 0.89 PV4395/1081574 Pass app Km FYN ZLYTE 85 85 85 0 Pass Pass0.7 P3042/1191778 Pass app RIPK2 LanthaScreen 84 86 85 1 Pass Pass 0.93PV4213/35334 Binding ACVR1 (ALK2) LanthaScreen 85 84 84 1 Pass Pass 0.92PV4877/676188 Binding Km MARK2 ZLYTE 86 82 84 4 Pass Pass 0.88PV3878/877056 Pass app KIT D816V y LanthaScreen 83 83 83 1 Pass Pass0.74 PV6199/1570986 Binding Km NUAK1 (ARK5) Adapta 83 83 83 0 Pass Pass0.83 PV4127/1401900 app Km PDGFRA V561D y ZLYTE 85 81 83 4 Pass Pass0.84 PV4680/1113217 Pass app TLK2 LanthaScreen 84 81 83 3 Pass Pass 0.91PV6424/1578677 Binding Km FLT3 D835Y y ZLYTE 83 81 82 2 Pass Pass 0.81PV3967/308809 Pass app Km PHKG2 ZLYTE 83 79 81 4 Pass Pass 0.85PV4555/37321 Pass app Km PDK1 Direct ZLYTE 85 74 79 11 Pass Pass 0.71P3001/1394674 Pass app Km CHEK1 (CHK1) ZLYTE 78 78 78 1 Pass Pass 0.74P3040/28702 Pass app DMPK LanthaScreen 76 79 78 2 Pass Pass 0.84PV3784/802854 Binding Km PAK7 (KIAA1264) ZLYTE 77 79 78 2 Pass Pass 0.84PV4405/36846 Pass app Km PLK3 ZLYTE 80 76 78 5 Pass Pass 0.8PV3812/38812 Pass app Km PDGFRA D842V y ZLYTE 77 73 75 4 Pass Pass 0.89PV4203/269691 Pass app Km JAK3 ZLYTE 73 73 73 1 Pass Pass 0.86PV3855/1017963 Pass app CDK16 LanthaScreen 70 73 71 3 Pass Pass 0.81PV6379/1577049 (PCTK1)/cyclin Y Binding Km MAP3K9 (MLK1) ZLYTE 68 73 705 Pass Pass 0.84 PV3787/1095726 Pass app Km NEK1 ZLYTE 68 70 69 3 PassPass 0.82 PV4202/880120 Pass app Km RET V804L y ZLYTE 71 66 69 5 PassPass 0.85 PV4397/36640 Pass app Km MINK1 ZLYTE 66 70 68 4 Pass Pass 0.76PV3810/1081579 Pass app ABL1 M351T y LanthaScreen 65 69 67 4 Pass Pass0.67 PV6151/1570962 Binding Km JAK2 ZLYTE 67 68 67 1 Pass Pass 0.8PV4210/784633 Pass app KIT N822K y LanthaScreen 66 67 66 1 Pass Pass0.71 PV6310/1576886 Binding Km TYK2 ZLYTE 65 64 64 1 Pass Pass 0.88PV4790/884908 Pass app FLT3 ITD LanthaScreen 64 62 63 2 Pass Pass 0.72PV6190/1570961 Binding Km MYLK2 ZLYTE 65 60 63 5 Pass Pass 0.67PV3757/36606 Pass app (skMLCK) 100 BRAF V599E y ZLYTE 63 61 62 2 PassPass 0.92 PV3849/910409 Pass Km FLT4 (VEGFR3) ZLYTE 64 60 62 5 Pass Pass0.87 PV4129/38454 Pass app Km PLK2 ZLYTE 60 64 62 4 Pass Pass 0.9PV4204/38798 Pass app Km ABL1 G250E y ZLYTE 60 61 60 1 Pass Pass 0.9PV3865/34529 Pass app ABL1 H396P y LanthaScreen 58 60 59 2 Pass Pass0.74 PV6148/1570966 Binding MAP3K10 (MLK2) LanthaScreen 58 60 59 2 PassPass 0.96 PV3877/1138344 Binding BMPR1B (ALK6) LanthaScreen 56 60 58 3Pass Pass 0.81 PV6235/1578462 Binding Km CHUK (IKK alpha) Adapta 65 5058 15 Pass Pass 0.79 PV4310/1110228 app DDR2 T654M y LanthaScreen 56 5757 1 Pass Pass 0.79 PV6175/1570973 Binding MAP4K1 (HPK1) LanthaScreen 5955 57 3 Pass Pass 0.72 PV6355/1575528 Binding MAPK8 (JNK1) LanthaScreen57 56 57 1 Pass Pass 0.83 PV3319/1354818 Binding Km SRC N1 ZLYTE 54 6057 6 Pass Pass 0.91 P2904/21068 Pass app STK33 LanthaScreen 59 55 57 4Pass Pass 0.91 PV4343/798867 Binding TGFBR1 (ALK5) LanthaScreen 56 58 572 Pass Pass 0.91 PV5837/562479 Binding DAPK2 LanthaScreen 60 53 56 7Pass Pass 0.64 PV3614/32159 Binding SIK1 LanthaScreen 57 54 56 2 PassPass 0.93 PV6445/1601253 Binding Km AMPK A1/B1/G1 ZLYTE 58 53 55 5 PassPass 0.9 PV4672/1046028 Pass app Km ABL1 Y253F y ZLYTE 61 48 54 12 PassPass 0.92 PV3863/34531 Pass app CDK14 LanthaScreen 56 51 54 5 Pass Pass0.56 PV6382/1577046 (PFTK1)/cyclin Y Binding DYRK2 LanthaScreen 54 53 541 Pass Pass 0.57 PV6331/1578676 Binding Km FGR ZLYTE 53 53 53 0 PassPass 0.94 P3041/26670 Pass app KIT Y823D y LanthaScreen 47 59 53 12 PassPass 0.67 PV6322/1579412 Binding Km LYN B ZLYTE 56 50 53 6 Pass Pass 0.8P2907/21076 Pass app NUAK2 LanthaScreen 54 52 53 1 Pass Pass 0.65PV6376/1607389 Binding TEK (TIE2) y LanthaScreen 53 52 53 0 Pass Pass0.6 PV6229/1570987 Y1108F Binding Km YES1 ZLYTE 55 51 53 4 Pass Pass0.84 A15557/50645 Pass app BRAF V599E y LanthaScreen 50 53 52 3 PassPass 0.89 PV3849/910409 Binding Km JAK2 JH1 JH2 ZLYTE 54 50 52 4 PassPass 0.84 PV4336/463344 Pass app V617F Km KDR (VEGFR2) ZLYTE 51 53 52 2Pass Pass 0.85 PV3660/1223246 Pass app Km ABL1 E255K y ZLYTE 53 50 51 3Pass Pass 0.79 PV3864/34528 Pass app Km AURKB (Aurora ZLYTE 54 48 51 6Pass Pass 0.78 PV6130/857013 Pass app B) BMPR1A (ALK3) LanthaScreen 5151 51 0 Pass Pass 0.82 PV6038/670004 Binding Km DAPK3 (ZIPK) ZLYTE 52 4951 3 Pass Pass 0.68 PV3686/1083962 Pass app PKN2 (PRK2) LanthaScreen 5152 51 2 Pass Pass 0.93 PV3879/555959 Binding Km RET Y791F y ZLYTE 57 4350 14 Pass Pass 0.91 PV4396/36639 Pass app STK38 (NDR) LanthaScreen 5149 50 2 Pass Pass 0.81 PV6370/1575531 Binding Km ABL1 ZLYTE 48 51 49 4Pass Pass 0.88 P3049/1012910 Pass app BMPR2 LanthaScreen 50 47 49 3 PassPass 0.8 PV6256/1579470 Binding MAPK10 (JNK3) LanthaScreen 48 49 49 1Pass Pass 0.93 PV4563/1075329 Binding Km LYN A ZLYTE 48 48 48 0 PassPass 0.91 P2906/827663 Pass app KIT V559D T6701 y LanthaScreen 46 49 473 Pass Pass 0.94 PV6316/1579472 Binding MET D1228H y LanthaScreen 46 4847 2 Pass Pass 0.78 PV6208/1570985 Binding ABL1 Q252H y LanthaScreen 5241 46 11 Pass Pass 0.76 PV6154/1570960 Binding AMPK (A1/B2/G1)LanthaScreen 49 43 46 5 Pass Pass 0.96 PV6244/1578463 Binding Km AMPKA2/B1/G1 ZLYTE 47 46 46 2 Pass Pass 0.93 PV4674/568101 Pass app BRAFLanthaScreen 44 47 46 2 Pass Pass 0.89 PV3848/1258788 Binding Km JAK2JH1 JH2 ZLYTE 46 47 46 1 Pass Pass 0.81 PV4393/311662 Pass app Km NEK9ZLYTE 46 45 46 1 Pass Pass 0.89 PV4653/38162 Pass app Km SYK ZLYTE 44 4946 5 Pass Pass 0.85 PV3857/756818 Pass app AMPK(A2/B2/G2) LanthaScreen44 45 45 1 Pass Pass 0.93 PV6250/1578465 Binding Km FLT3 ZLYTE 42 49 457 Pass Pass 0.78 PV3182/1012909 Pass app Km IKBKB (IKK beta) ZLYTE 52 3945 13 Pass Pass 0.9 PV3836/1445179 Pass app Km SRC ZLYTE 45 44 45 1 PassPass 0.89 P3044/1255538 Pass app TNIK LanthaScreen 46 43 45 3 Pass Pass0.8 PV6427/1578672 Binding Km RET ZLYTE 46 42 44 4 Pass Pass 0.89PV3819/853376 Pass app TAOK1 LanthaScreen 45 43 44 2 Pass Pass 0.82PV6415/1576240 Binding Km DAPK1 Adapta 42 43 43 2 Pass Pass 0.83PV3969/32654 app Km PDGFRA (PDGFR ZLYTE 43 43 43 0 Pass Pass 0.82PV3811/1269727 Pass app alpha) Km BLK ZLYTE 41 42 42 1 Pass Pass 0.8PV3683/33635 Pass app CDK2/cyclin O LanthaScreen 39 42 41 4 Pass Pass0.75 PV6286/1576891 Binding TEK (TIE2) y LanthaScreen 40 41 41 1 PassPass 0.92 PV6226/1570990 R849W Binding Km TYRO3 (RSE) ZLYTE 42 39 41 4Pass Pass 0.9 PV3828/682475 Pass app Km ABL2 (Arg) ZLYTE 40 39 40 1 PassPass 0.92 PV3266/850069 Pass app AMPK (A1/B1/G2) LanthaScreen 40 40 40 0Pass Pass 0.97 PV6238/1578461 Binding Km LCK ZLYTE 41 38 40 3 Pass Pass0.79 P3043/850070 Pass app LIMK1 LanthaScreen 40 39 40 1 Pass Pass 0.87PV4337/367810 Binding AMPK (A1/B1/G3) LanthaScreen 37 42 39 5 Pass Pass0.92 PV6241/1578466 Binding Km MST1R (RON) ZLYTE 38 38 38 0 Pass Pass0.83 PV4314/765277 Pass app Km PHKG1 ZLYTE 39 37 38 2 Pass Pass 0.75PV3853/830447 Pass app Km CDK2/cyclin A ZLYTE 38 36 37 1 Pass Pass 0.88PV3267/924344 Pass app DDR2 N456S y LanthaScreen 33 42 37 9 Pass Pass0.86 PV6172/1570968 Binding Km RPS6KB1 ZLYTE 37 36 37 1 Pass Pass 0.84PV3815/38944 Pass app (p70S6K) CDK2/cyclin A1 LanthaScreen 33 39 36 6Pass Pass 0.89 PV6289/1576888 Binding Km PDGFRA T674I y ZLYTE 40 32 36 8Pass Pass 0.67 PV3847/35891 Pass app ACVRL1 (ALK1) LanthaScreen 33 38 355 Pass Pass 0.79 PV4883/511550 Binding AMPK (A2/B2/G1) LanthaScreen 3535 35 0 Pass Pass 0.94 PV6247/1578460 Binding FGFR3 K650M y LanthaScreen33 38 35 5 Pass Pass 0.82 PV6187/1573650 Binding Km ITK ZLYTE 37 32 34 4Pass Pass 0.71 PV3875/1430640 Pass app STK38L (NDR2) LanthaScreen 30 3834 9 Pass Pass 0.54 PV6373/1575530 Binding Km CLK1 ZLYTE 33 34 33 0 PassPass 0.88 PV3315/943590 Pass app Km EPHB1 ZLYTE 34 31 33 3 Pass Pass0.91 PV3786/34225 Pass app RET V804M y LanthaScreen 35 31 33 4 Pass Pass0.82 PV6223/1570988 Binding TTK LanthaScreen 33 34 33 1 Pass Pass 0.77PV3792/558743 Binding Km PDGFRB (PDGFR ZLYTE 34 30 32 4 Pass Pass 0.78P3082/27567 Pass app beta) WEE1 LanthaScreen 28 36 32 7 Pass Pass 0.64PV3817/784632 Binding Km CSNK2A2 (CK2 ZLYTE 32 30 31 2 Pass Pass 0.81PV3624/32653 Pass app alpha 2) Km EGFR (ErbB1) y ZLYTE 34 29 31 4 PassPass 0.9 PV4879/1189155 Pass app T790M L858R Km MET (cMet) ZLYTE 30 3331 4 Pass Pass 0.79 PV3143/625156 Pass app Km TXK ZLYTE 27 36 31 9 PassPass 0.86 PV5860/750657 Pass app Km MET M1250T y ZLYTE 26 34 30 8 PassPass 0.86 PV3968/34718 Pass app Km NEK2 ZLYTE 29 30 30 0 Pass Pass 0.81PV3360/1086245 Pass app SLK LanthaScreen 29 32 30 4 Pass Pass 0.95PV3830/34390 Binding Km ABL1 T315I y ZLYTE 30 29 29 1 Pass Pass 0.84PV3866/39639 Pass app ACVR2B LanthaScreen 27 31 29 4 Pass Pass 0.86PV6049/877066 Binding Km DYRK1A ZLYTE 30 27 28 4 Pass Pass 0.9PV3785/683159 Pass app NLK LanthaScreen 26 29 28 3 Pass Pass 0.88PV4309/35323 Binding Km ROS1 ZLYTE 30 26 28 4 Pass Pass 0.81PV3814/479684 Pass app AXL R499C y LanthaScreen 28 27 27 1 Pass Pass0.89 PV6253/1578673 Binding Km CHEK2 (CHK2) ZLYTE 27 27 27 0 Pass Pass0.84 PV3367/1033750 Pass app Km EPHA1 ZLYTE 29 24 27 5 Pass Pass 0.85PV3841/1138343 Pass app Km HCK ZLYTE 26 28 27 2 Pass Pass 0.87PV6128/862448 Pass app Km SGK (SGK1) ZLYTE 28 26 27 2 Pass Pass 0.88PV3818/1088196 Pass app CDK2/cyclin A2 LanthaScreen 24 29 26 5 Pass Pass0.7 PV6292/1576892 Binding MAP3K11 (MLK3) LanthaScreen 24 29 26 5 PassPass 0.93 PV3788/869925 Binding MERTK (cMER) y LanthaScreen 24 27 26 3Pass Pass 0.92 PV6325/1578675 A708S Binding Km PRKX ZLYTE 22 30 26 8Pass Pass 0.87 PV3813/34283 Pass app Km DYRK3 ZLYTE 26 24 25 2 Pass Pass0.89 PV3837/290370 Pass app Km EGFR (ErbB1) y ZLYTE 25 25 25 0 Pass Pass0.85 PV4803/1123633 Pass app T790M 100 MAPK8 (JNK1) ZLYTE 33 18 25 15Pass Pass 0.79 PV3319/1354818 Pass TNK2 (ACK) LanthaScreen 25 26 25 1Pass Pass 0.92 PV4807/407338 Binding Km AXL ZLYTE 22 25 24 3 Pass Pass0.86 PV3971/873922 Pass app LATS2 LanthaScreen 23 25 24 1 Pass Pass 0.91PV6364/1575533 Binding Km MERTK (cMER) ZLYTE 27 22 24 5 Pass Pass 0.82PV3627/32658 Pass app PRKACG (PRKAC LanthaScreen 28 19 24 9 Pass Pass0.66 PV6391/1577047 gamma) Binding Km AURKC (Aurora ZLYTE 23 23 23 0Pass Pass 0.66 PV3856/1078206 Pass app C) FGFR1 V561M y LanthaScreen 2025 23 5 Pass Pass 0.91 PV6343/1578678 Binding Km MELK ZLYTE 25 22 23 3Pass Pass 0.85 PV4823/819467 Pass app Km NEK4 ZLYTE 23 23 23 0 Pass Pass0.88 PV4315/924342 Pass app ACVR2A LanthaScreen 23 21 22 2 Pass Pass0.76 PV6124/862446 Binding ICK LanthaScreen 22 22 22 0 Pass Pass 0.86PV6358/1577056 Binding MAP3K14 (NIK) LanthaScreen 20 24 22 3 Pass Pass0.91 PV4902/1296958 Binding MAP3K7/MAP3K7 LanthaScreen 22 22 22 0 PassPass 0.89 PV4394/930475 IP1 (TAK1-TAB1) Binding Km GSK3A (GSK3 ZLYTE 2022 21 2 Pass Pass 0.91 PV6126/862449 Pass app alpha) Km GSK3B (GSK3ZLYTE 21 21 21 0 Pass Pass 0.79 PV3365/371501 Pass app beta) KmCDK9/cyclin T1 Adapta 18 22 20 4 Pass Pass 0.88 PV4131/1370615 app KmEPHA4 ZLYTE 18 21 20 2 Pass Pass 0.9 PV3651/32933 Pass app Km HIPK2ZLYTE 21 19 20 2 Pass Pass 0.93 PV5275/452552 Pass app 100 MAPK10 (JNK3)ZLYTE 20 21 20 0 Pass Pass 0.9 PV4563/1075329 Pass Km CAMK2D ZLYTE 18 1919 1 Pass Pass 0.91 PV3373/31647 Pass app (CaMKII delta) CAMK2GLanthaScreen 18 19 18 1 Pass Pass 0.77 PV6268/1576884 (CaMKII gamma)Binding FYN A LanthaScreen 16 21 18 6 Pass Pass 0.81 PV6346/1575529Binding KIT D820E y LanthaScreen 18 19 18 2 Pass Pass 0.97PV6307/1576885 Binding MAP4K3 (GLK) LanthaScreen 16 21 18 6 Pass Pass0.87 PV6349/1579471 Binding MYO3B (MYO3 LanthaScreen 17 20 18 3 PassPass 0.95 PV6367/1577055 beta) Binding Km NTRK1 (TRKA) ZLYTE 16 20 18 3Pass Pass 0.73 PV3144/1347534 Pass app Km NTRK3 (TRKC) ZLYTE 17 19 18 3Pass Pass 0.85 PV3617/708766 Pass app RAF1 (cRAF) y LanthaScreen 15 2018 4 Pass Pass 0.91 PV3805/1293604 Y340D Y341D Binding RET G691S yLanthaScreen 19 18 18 1 Pass Pass 0.8 PV6214/1570982 Binding BRSK2LanthaScreen 16 17 17 1 Pass Pass 0.87 PV6259/1576239 Binding LIMK2LanthaScreen 14 19 17 5 Pass Pass 0.95 PV3860/355434 Binding PRKACB(PRKAC LanthaScreen 16 18 17 2 Pass Pass 0.82 PV6388/1577048 beta)Binding Km CDK1/cyclin B ZLYTE 16 15 16 1 Pass Pass 0.85 PV3292/873341Pass app CDK2/cyclin E1 LanthaScreen 15 17 16 2 Pass Pass 0.66PV6295/1576887 Binding Km EPHB2 ZLYTE 14 17 16 3 Pass Pass 0.91PV3625/1386867 Pass app Km HIPK4 ZLYTE 18 14 16 4 Pass Pass 0.89PV3852/719847 Pass app 100 MAPK9 (JNK2) ZLYTE 12 21 16 9 Pass Pass 0.73PV3620/32388 Pass Km PIK3CD/PIK3R1 Adapta 22 9 16 12 Pass Pass 0.88PV5273/1147693 app (p110 de1ta/p85 alpha) Km RPS6KA6 (RSK4) ZLYTE 14 1716 3 Pass Pass 0.63 PV4557/37496 Pass app Km CDK7/cyclin Adapta 17 14 152 Pass Pass 0.8 PV3868/1427412 app H/MNAT1 Km EPHB4 ZLYTE 15 14 15 2Pass Pass 0.73 PV3251/29241 Pass app 100 MAP3K8 (COT) ZLYTE 19 11 15 7Pass Pass 0.89 PV4313/1111069 Pass 100 PDK1 ZLYTE 14 16 15 1 Pass Pass0.81 P3001/1394674 Pass Km RPS6KA2 (RSK3) ZLYTE 17 13 15 4 Pass Pass0.89 PV3846/34468 Pass app TLK1 LanthaScreen 13 17 15 4 Pass Pass 0.88PV6421/1576241 Binding EGFR (ErbB1) y LanthaScreen 14 13 14 1 Pass Pass0.78 PV6178/1570967 d746-750 Binding EPHA6 LanthaScreen 9 20 14 11 PassPass 0.63 PV6337/1575527 Binding Km PRKG2 (PKG2) ZLYTE 16 13 14 3 PassPass 0.87 PV3973/273926 Pass app Km RPS6KA3 (RSK2) ZLYTE 17 10 14 7 PassPass 0.83 PV3323/1361669 Pass app Km TEK (Tie2) ZLYTE 11 17 14 6 PassPass 0.59 PV3628/34398 Pass app ALK F1174L Y LanthaScreen 12 14 13 2Pass Pass 0.91 PV6160/1570964 Binding Km HIPK3 (YAK1) ZLYTE 15 11 13 4Pass Pass 0.92 PV4209/1076022 Pass app MAPK9 (JNK2) LanthaScreen 12 1413 3 Pass Pass 0.91 PV3620/32388 Binding Km ROCK2 ZLYTE 12 14 13 2 PassPass 0.74 PV3759/843703 Pass app Km RPS6KA1 (RSK1) ZLYTE 15 11 13 4 PassPass 0.83 PV3680/880119 Pass app Km STK22B (TSSK2 ) ZLYTE 12 14 13 1Pass Pass 0.79 PV3622/32396 Pass app Km BMX ZLYTE 12 13 12 1 Pass Pass0.89 PV3371/953336 Pass app Km CAMK1D (CaMKl ZLYTE 12 13 12 1 Pass Pass0.85 PV3663/1042984 Pass app delta) Km CSK ZLYTE 13 10 12 2 Pass Pass0.88 P2927/1205898 Pass app Km FGFR2 ZLYTE 12 11 12 1 Pass Pass 0.93PV3368/31517 Pass app Km FRK (PTK5) ZLYTE 11 13 12 2 Pass Pass 0.84PV3874/34553 Pass app Km HIPK1 (Myak) ZLYTE 11 13 12 1 Pass Pass 0.89PV4561/1126221 Pass app MAP3K3 LanthaScreen 10 13 12 3 Pass Pass 0.79PV3876/702480 (MEKK3) Binding Km MAP4K5 (KHS1) ZLYTE 11 12 12 0 PassPass 0.65 PV3682/1383139 Pass app Km MUSK ZLYTE 15 8 12 7 Pass Pass 0.67PV3834/1217900 Pass app RET M918T y LanthaScreen 10 13 12 3 Pass Pass0.64 PV6217/1570989 Binding Km DYRK1B ZLYTE 14 9 11 5 Pass Pass 0.91PV4649/877059 Pass app EPHA7 LanthaScreen 10 12 11 2 Pass Pass 0.62PV3689/33790 Binding Km PLK1 ZLYTE 12 9 11 3 Pass Pass 0.89 PV3501/39441Pass app Km CAMK2B ZLYTE 11 8 10 3 Pass Pass 0.81 PV4205/35330 Pass app(CaMKII beta) Km CDC42 BPB ZLYTE 6 13 10 7 Pass Pass 0.88 PV4399/36845Pass app (MRCKB) CDK9 (Inactive) LanthaScreen 8 13 10 5 Pass Pass 0.93PV6304/1579469 Binding CDK9/cyclin K LanthaScreen 11 9 10 2 Pass Pass0.74 PV4335/35774 Binding Km CSNK2A1 (CK2 ZLYTE 13 7 10 7 Pass Pass 0.73PV3248/1240448 Pass app alpha 1) Km JAK1 ZLYTE 11 10 10 2 Pass Pass 0.62PV4774/1240449 Pass app MAP3K2 LanthaScreen 11 9 10 2 Pass Pass 0.75PV3822/1171755 (MEKK2) Binding Km PRKCB2 (PKC ZLYTE 12 8 10 4 Pass Pass0.82 P2251/930444 Pass app beta II) Km PRKCH (PKC eta) ZLYTE 12 8 10 4Pass Pass 0.62 P2633/25587 Pass app Km ROCK1 ZLYTE 6 14 10 8 Pass Pass0.78 PV3691/37178 Pass app Km SGK2 ZLYTE 7 12 10 5 Pass Pass 0.78PV3858/1099019 Pass app STK39 (STLK3) LanthaScreen 10 9 10 1 Pass Pass0.54 PV6412/1608283 Binding ALK C1156Y y LanthaScreen 7 12 9 5 Pass Pass0.98 PV6157/1570963 Binding Km EGFR (ErbB1) ZLYTE 10 9 9 2 Pass Pass0.89 PV3872/1004026 Pass app Km EPHA8 ZLYTE 11 7 9 4 Pass Pass 0.81PV3844/36870 Pass app KIT V654A y LanthaScreen 7 10 9 3 Pass Pass 0.82PV4132/35129 Binding 100 MAP2K6 (MKK6) ZLYTE 0 19 9 19 Pass Pass 0.85PV3318/884909 Pass Km NEK6 ZLYTE 12 6 9 7 Pass Pass 0.76 PV3353/30778Pass app Km PIK3CG (p110) Adapta 11 8 9 3 Pass Pass 0.92 PV4786/1153861app gamma) CAMKK2 LanthaScreen 7 9 8 2 Pass Pass 0.93 PV4206/35319(CaMKK beta) Binding CASK LanthaScreen 6 10 8 4 Pass Pass 0.75PV6271/1576243 Binding Km FES (FPS) ZLYTE 9 7 8 2 Pass Pass 0.84PV3354/35734 Pass app FGFR3 G697C y LanthaScreen 13 4 8 9 Pass Pass 0.74PV6184/1570969 Binding Km FGFR3 K650E y ZLYTE 11 6 8 5 Pass Pass 0.85PV4392/36445 Pass app MAP2K1 (MEK1) y LanthaScreen 10 7 8 2 Pass Pass0.92 P3099/38541 S218D S222D Binding 100 MAP2K2 (MEK2) ZLYTE 13 2 8 10Pass Pass 0.92 PV3615/32519 Pass Km MAPK13 (p38 ZLYTE 7 9 8 2 Pass Pass0.83 PV3656/36817 Pass app delta) Km PAK1 ZLYTE 8 8 8 0 Pass Pass 0.86PV3820/35463 Pass app ZAK LanthaScreen 6 10 8 4 Pass Pass 0.92PV3882/34603 Binding  10 CAMK1 (CaMK1) Adapta 7 8 7 1 Pass Pass 0.86PV4391/36046 CDK1/cyclin A2 LanthaScreen 2 12 7 11 Pass Pass 0.73PV6280/1579468 Binding Km CLK2 ZLYTE 5 9 7 4 Pass Pass 0.81PV4201/873335 Pass app Km DCAMKL2 ZLYTE 11 3 7 8 Pass Pass 0.63PV4297/869931 Pass app (DCK2) Km FER ZLYTE 4 9 7 5 Pass Pass 0.71PV3806/38946 Pass app Km FRAP1 (mTOR) ZLYTE 2 12 7 10 Pass Pass 0.8PV4753/873345 Pass app MAP2K3 (MEK3) LanthaScreen 5 8 7 4 Pass Pass 0.61PV3662/357368 Binding MAP3K5 (ASK1) LanthaScreen 7 8 7 1 Pass Pass 0.72PV3809/666419 Binding Km PIM1 ZLYTE 11 3 7 9 Pass Pass 0.76PV3503/811382 Pass app CDK3/cyclin E1 LanthaScreen 2 9 6 7 Pass Pass0.62 PV6298/1579411 Binding Km EPHA2 ZLYTE 1 12 6 12 Pass Pass 0.77PV3688/36904 Pass app Km ERBB4 (HER4) ZLYTE 8 5 6 4 Pass Pass 0.74PV3626/32657 Pass app Km FLT1 (VEGFR1) ZLYTE 7 4 6 3 Pass Pass 0.81PV3666/33924 Pass app Km IRAK4 ZLYTE 5 8 6 3 Pass Pass 0.78PV3362/1088346 Pass app Km KIT T670I y ZLYTE 6 7 6 2 Pass Pass 0.71PV3869/34504 Pass app Km LTK (TYK1) ZLYTE 5 7 6 1 Pass Pass 0.78PV4651/768522 Pass app Km MAPK1 (ERK2) ZLYTE 6 6 6 0 Pass Pass 0.86PV3313/904347 Pass app Km MAPKAPK5 ZLYTE 6 7 6 1 Pass Pass 0.78PV3301/880117 Pass app (PRAK) Km NTRK2 (TRKB) ZLYTE 5 7 6 1 Pass Pass0.93 PV3616/35706 Pass app Km PAK6 ZLYTE 9 4 6 5 Pass Pass 0.8PV3502/625425 Pass app Km PRKCI (PKC iota) ZLYTE 5 7 6 2 Pass Pass 0.77PV3183/28662 Pass app Km PTK2B (FAK2) ZLYTE 8 4 6 4 Pass Pass 0.8PV4567/883370 Pass app TESK2 LanthaScreen 14 −3 6 16 Pass Pass 0.74PV6418/1576242 Binding Km ADRBK1 (GRK2) ZLYTE 3 7 5 4 Pass Pass 0.88PV3361/883372 Pass app Km ADRBK2 (GRK3) ZLYTE 6 4 5 2 Pass Pass 0.71PV3827/38897 Pass app ALK L1196M y LanthaScreen 1 9 5 8 Pass Pass 0.92PV6166/1570971 Binding Km BTK ZLYTE 3 6 5 3 Pass Pass 0.89PV3363/1405298 Pass app Km CDK5/p35 ZLYTE 7 2 5 5 Pass Pass 0.89PV3000/25348 Pass app Km EGFR (ErbB1) y ZLYTE 6 5 5 1 Pass Pass 0.89PV3873/34562 Pass app L861Q Km FGFR3 ZLYTE 6 4 5 2 Pass Pass 0.84PV3145/28459 Pass app GRK1 LanthaScreen 7 2 5 5 Pass Pass 0.96PV6352/1577053 Binding 100 MAP2K1 (MEK1) ZLYTE 7 3 5 4 Pass Pass 0.82PV3303/1081576 Pass MAP2K1 (MEK1) LanthaScreen 6 5 5 1 Pass Pass 0.95PV3303/1081567 Binding MAP2K2 (MEK2) LanthaScreen 5 5 5 0 Pass Pass 0.94PV3615/32519 Binding MAP2K6 (MKK6) y LanthaScreen 5 5 5 0 Pass Pass 0.91PV3293/877061 S207E T211E Binding Km MAPK11 (p38 ZLYTE 6 4 5 2 Pass Pass0.85 PV3679/1131827 Pass app beta) Km PAK3 ZLYTE 7 2 5 4 Pass Pass 0.86PV3789/34118 Pass app Km PI4KB (PI4K beta) Adapta 7 3 5 4 Pass Pass 0.95PV5277/943589 app Km STK3 (MST2) ZLYTE 5 6 5 1 Pass Pass 0.7PV4805/371195 Pass app STK32B (YANK2) LanthaScreen 1 8 5 7 Pass Pass 0.9PV6406/1577058 Binding Km TAOK2 (TAO1) ZLYTE 3 6 5 3 Pass Pass 0.8PV3760/1011094 Pass app ALK R1275Q y LanthaScreen 0 7 4 7 Pass Pass 0.89PV6169/1570970 Binding Km CDC42 BPA ZLYTE 4 4 4 0 Pass Pass 0.7PV4398/1314130 Pass app (MRCKA) Km EGFR (ErbB1) y ZLYTE 6 3 4 3 PassPass 0.72 PV4128/279551 Pass app L858R EPHA3 LanthaScreen 6 3 4 4 PassPass 0.82 PV3359/673524 Binding Km GRK7 ZLYTE 3 5 4 2 Pass Pass 0.86PV3823/34013 Pass app Km INSRR (IRR) ZLYTE 5 4 4 1 Pass Pass 0.91PV3808/34272 Pass app 100 MAPK14 (p38 ZLYTE −1 9 4 10 Pass Pass 0.87PV3304/1475037 Pass alpha) MYLK (MLCK) LanthaScreen 5 3 4 2 Pass Pass0.91 PV4339/36152 Binding Km PRKCB1 (PKC ZLYTE 5 3 4 2 Pass Pass 0.71P2291/299686 Pass app beta 1) 100 RAF1 (cRAF) y ZLYTE 5 3 4 2 Pass Pass0.91 PV3805/1293604 Pass Y340D Y341D Km RPS6KA5 (MSK1) ZLYTE 6 3 4 2Pass Pass 0.84 PV3681/380935 Pass app Km SRMS (Srm) ZLYTE 5 3 4 2 PassPass 0.84 PV4214/1110226 Pass app Km ZAP70 ZLYTE 4 4 4 0 Pass Pass 0.86P2782/843705 Pass app Km AKT2 (PKB beta) ZLYTE 3 2 3 1 Pass Pass 0.81PV3184/28770 Pass app Km EPHA5 ZLYTE 3 4 3 1 Pass Pass 0.74 PV3840/34383Pass app Km EPHB3 ZLYTE 3 3 3 0 Pass Pass 0.94 PV3658/33066 Pass app KmFGFR4 ZLYTE 7 −2 3 9 Pass Pass 0.65 P3054/26967 Pass app Km KIT ZLYTE 15 3 4 Pass Pass 0.76 P3081/1344384 Pass app Km MAPK12 (p38 ZLYTE 4 3 3 1Pass Pass 0.87 PV3654/1140849 Pass app gamma) Km NEK7 ZLYTE 5 2 3 3 PassPass 0.81 PV3833/34387 Pass app Km PAK2 (PAK65) ZLYTE 6 −1 3 7 Pass Pass0.87 PV4565/924347 Pass app Km PIM2 ZLYTE −1 7 3 7 Pass Pass 0.76PV3649/32930 Pass app Km PRKACA (PKA) ZLYTE 4 1 3 3 Pass Pass 0.8P2912/37377 Pass app Km PRKG1 ZLYTE 2 5 3 3 Pass Pass 0.9 PV4340/893283Pass app Km SGKL (SGK3) ZLYTE 3 4 3 1 Pass Pass 0.87 PV3859/38954 Passapp Km AKT1 (PKB alpha) ZLYTE 1 3 2 2 Pass Pass 0.89 P2999/1159806 Passapp Km AKT3 (PKB ZLYTE 3 1 2 2 Pass Pass 0.88 PV3185/28771 Pass appgamma) Km CAMK2A ZLYTE 4 0 2 4 Pass Pass 0.89 PV3142/28192 Pass app(CaMKII alpha) Km CDK5/p25 ZLYTE 3 1 2 2 Pass Pass 0.79 PV4676/907645Pass app Km CLK3 ZLYTE 1 3 2 1 Pass Pass 0.88 PV3826/939820 Pass appDDR1 LanthaScreen 3 2 2 1 Pass Pass 0.95 PV6047/693053 Binding Km GRK6ZLYTE 2 2 2 1 Pass Pass 0.84 PV3661/37437 Pass app KIT T670E yLanthaScreen 2 2 2 1 Pass Pass 0.9 PV6313/1575536 Binding Km MAP4K2(GCK) ZLYTE 6 −2 2 8 Pass Pass 0.88 PV4211/748356 Pass app Km MATK (HYL)ZLYTE 1 2 2 0 Pass Pass 0.83 PV3370/31553 Pass app Km MST4 ZLYTE 2 3 2 0Pass Pass 0.76 PV3690/1205875 Pass app Km PASK ZLYTE 3 1 2 2 Pass Pass0.68 PV3972/762487 Pass app Km STK23 (MSSK1) ZLYTE 2 1 2 1 Pass Pass0.86 PV3880/1214750 Pass app TAOK3 (JIK) LanthaScreen 3 1 2 2 Pass Pass0.86 PV3652/32935 Binding Km ALK ZLYTE 4 −2 1 5 Pass Pass 0.86PV3867/1542512 Pass app CDK8/cyclin C LanthaScreen 0 3 1 4 Pass Pass0.94 PV4402/1177216 Binding Km CSNK1A1 (CK1 ZLYTE 2 1 1 1 Pass Pass 0.84PV3850/1004025 Pass app alpha 1) Km CSNK1E (CK1 ZLYTE −2 4 1 6 Pass Pass0.75 PV3500/866725 Pass app epsilon) Km CSNK1G3 (CK1 ZLYTE 4 −2 1 6 PassPass 0.85 PV3838/1140848 Pass app gamma 3) Km IGF1R ZLYTE 1 2 1 2 PassPass 0.78 PV3250/924345 Pass app MAP2K6 (MKK6) LanthaScreen −2 4 1 6Pass Pass 0.86 PV3318/884909 Binding Km MAPK14 (p38 ZLYTE 2 1 1 1 PassPass 0.94 PV3304/1475037 Pass app alpha) Direct Km PTK2 (FAK) ZLYTE 0 21 2 Pass Pass 0.79 PV3832/1378055 Pass app Km RPS6KA4 (MSK2) ZLYTE 0 2 13 Pass Pass 0.84 PV3782/990109 Pass app Km STK24 (MST3) ZLYTE −5 8 1 14Pass Pass 0.7 PV3650/32932 Pass app TEC LanthaScreen 0 2 1 2 Pass Pass0.93 PV3269/910411 Binding WNK2 LanthaScreen −4 5 1 9 Pass Pass 0.86PV4341/35976 Binding CAMKK1 LanthaScreen 2 −1 0 2 Pass Pass 0.8PV4670/406782 (CAMKKA) Binding DDR2 LanthaScreen 0 1 0 2 Pass Pass 0.94PV3870/916220 Binding Km 1NSR ZLYTE −1 1 0 3 Pass Pass 0.81PV3781/1314127 Pass app Km MAPK3 (ERK1) ZLYTE 3 −2 0 5 Pass Pass 0.86PV3311/1255534 Pass app Km MAPKAPK2 ZLYTE 0 −1 0 1 Pass Pass 0.82PV3317/36559 Pass app  10 PI4KA (PI4K Adapta 3 −3 0 6 Pass Pass 0.81PV5689/1131829 alpha) Km PRKCD (PKC ZLYTE 4 −4 0 8 Pass Pass 0.73P2293/39038 Pass app delta) RIPK3 LanthaScreen −1 1 0 2 Pass Pass 0.84PV6397/1610742 Binding Km SPHK1 Adapta 10 −9 0 19 Pass Pass 0.77PV5214/933639 app Km STK4 (MST1) ZLYTE 0 0 0 0 Pass Pass 0.73PV3854/38395 Pass app CDK5 (Inactive) LanthaScreen 1 −2 −1 3 Pass Pass0.68 PV6301/1576893 Binding Km DNA-PK ZLYTE 2 −4 −1 7 Pass Pass 0.69PV5864/1594760 Pass app Km DYRK4 ZLYTE −2 0 −1 1 Pass Pass 0.82PV3871/37361 Pass app Km EEF2K ZLYTE −1 0 −1 1 Pass Pass 0.87PV4559/1075327 Pass app Km ERBB2 (HER2) ZLYTE 0 −3 −1 3 Pass Pass 0.74PV3366/1185123 Pass app Km PTK6 (Brk) ZLYTE −1 −1 −1 0 Pass Pass 0.86PV3291/1205876 Pass app STK32C (YANK3) LanthaScreen −4 3 −1 6 Pass Pass0.68 PV6409/1577045 Binding Km CAMK4 ZLYTE −2 −2 −2 0 Pass Pass 0.84PV3310/1103512 Pass app (CaMKIV ) LATS1 LanthaScreen −5 1 −2 6 Pass Pass0.62 PV6361/1575532 Binding  10 PIK3C2B (PI3K- Adapta 3 −7 −2 9 PassPass 0.87 PV5374/1223244 C2 beta) Km PRKCA (PKC ZLYTE 0 −5 −2 6 PassPass 0.71 P2232/38479 Pass app alpha) Km SRPK1 ZLYTE −1 −2 −2 2 PassPass 0.94 PV4215/1182336 Pass app Km STK25 (YSK1) ZLYTE 0 −4 −2 4 PassPass 0.8 PV3657/33163 Pass app WNK3 LanthaScreen −3 0 −2 2 Pass Pass0.83 PV4342/36047 Binding Km CSNK1D (CK1 ZLYTE −1 −5 −3 5 Pass Pass 0.83PV3665/843704 Pass app delta) Km FGFR1 ZLYTE −5 −2 −3 3 Pass Pass 0.8PV3146/28427 Pass app Km GRK5 ZLYTE 5 −10 −3 15 Pass Pass 0.82PV3824/879275 Pass app Km MAPKAPK3 ZLYTE 1 −8 −3 9 Pass Pass 0.86PV3299/38895 Pass app Km PRKCE (PKC ZLYTE 1 −6 −3 8 Pass Pass 0.83P2292/37717 Pass app epsilon) Km SRPK2 ZLYTE −2 −4 −3 2 Pass Pass 0.91PV3829/900365 Pass app CDK11 (Inactive) LanthaScreen −1 −7 −4 7 PassPass 0.71 PV6283/1576889 Binding Km GRK4 ZLYTE −4 −5 −5 1 Pass Pass 0.86PV3807/618977 Pass app Km PIK3CA/PIK3R1 Adapta −5 −4 −5 1 Pass Pass 0.79PV4788/616250 app (p110 alpha/p85 alpha) Km PKN1 (PRK1) ZLYTE −6 −4 −5 2Pass Pass 0.68 PV3790/356552 Pass app Km PRKCG (PKC ZLYTE −9 −1 −5 8Pass Pass 0.73 P2233/39126 Pass app gamma) Km CSNK1G1 (CK1 ZLYTE −14 2−6 16 Pass Pass 0.77 PV3825/34360 Pass app gamma 1) Km GSG2 (Haspin)Adapta 7 −20 −6 26 Pass Pass 0.71 PV5708/869949 app Km ACVR1B (ALK4)ZLYTE −10 −4 −7 6 Pass Pass 0.9 PV4312/919690 Pass app Km PIK3C3(hVPS34) Adapta −5 −9 −7 4 Pass Pass 0.91 PV5126/853378 app Km PIK3C2A(PI3K- Adapta −7 −9 −8 2 Pass Pass 0.88 PV5586/1123632 app C2 alpha)  10SPHK2 Adapta −16 −2 −9 14 Pass Pass 0.56 PV5216/1296957 Km CSNK1G2 (CK1ZLYTE −7 −16 −11 9 Pass Pass 0.79 PV3499/1120155 Pass app gamma 2) KmPRKCQ (PKC ZLYTE −6 −17 −12 11 Pass Pass 0.76 P2996/26231 Pass apptheta) 100 BRAF ZLYTE −18 −10 −14 8 Pass Pass 0.83 PV3848/1258788 PassKm PRKCZ (PKC zeta) ZLYTE −18 −16 −17 2 Pass Pass 0.7 P2273/31602 Passapp

TABLE S5-2 fold selectivity fold selectivity Technology ATP IC50 (nM)Hillslope R2 (TBK1) (IKBKE) ZLYTE Km app 1.04 1.12 0.9966 1.0 0.2LanthaScreen 2.71 0.8 0.9917 2.6 0.5 Binding LanthaScreen 4.35 1.270.9998 4.2 0.8 Binding ZLYTE Km app 5.59 1.06 0.9955 5.4 1.0 ZLYTE Kmapp 14.2 0.55 0.9914 13.7 2.5 LanthaScreen 14.8 1.25 0.9989 14.2 2.6Binding LanthaScreen 30.9 0.84 0.9797 29.7 5.5 Binding LanthaScreen 31.50.88 0.9956 30.3 5.6 Binding Adapta Km app 34.9 0.99 0.9945 33.6 6.2ZLYTE Km app 35.1 1 0.9994 33.8 6.3 ZLYTE Km app 35.8 1.11 0.9993 34.46.4 ZLYTE Km app 41.5 1.49 0.9978 39.9 7.4 ZLYTE Km app 45.6 1.01 0.998143.8 8.2 ZLYTE Km app 47.7 0.84 0.9995 45.9 8.5 ZLYTE Km app 55 0.960.9993 52.9 9.8 ZLYTE Km app 59 0.69 0.9971 56.7 10.6 LanthaScreen 74.11 0.9972 71.3 13.3 Binding LanthaScreen 78.7 0.86 0.9971 75.7 14.1Binding Adapta Km app 80.6 1 0.9983 77.5 14.4 ZLYTE Km app 81.3 1.120.9991 78.2 14.5 LanthaScreen 81.6 1.42 0.9789 78.5 14.6 Binding ZLYTEKm app 90.9 1.11 0.9974 87.4 16.3 LanthaScreen 104 1.22 0.997 100.0 18.6Binding LanthaScreen 156 0.95 0.9986 150.0 27.9 Binding

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EQUIVALENTS AND SCOPE

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents, and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an.” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined. i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements): etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of.” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one. B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03. It should be appreciatedthat embodiments described in this document using an open-endedtransitional phrase (e.g., “comprising”) are also contemplated, inalternative embodiments, as “consisting of” and “consisting essentiallyof” the feature described by the open-ended transitional phrase. Forexample, if the disclosure describes “a composition comprising A and B,”the disclosure also contemplates the alternative embodiments “acomposition consisting of A and B” and “a composition consistingessentially of A and B.”

What is claimed is:
 1. A method for evaluating tumor cell spheroids in athree-dimensional microfluidic device, the method comprising: obtainingtumor spheroids from an enzyme treated tumor sample, suspending a firstaliquot of the tumor spheroids in biocompatible gel; suspending a secondaliquot of the tumor spheroids in biocompatible gel; placing the firstaliquot of the tumor spheroids in biocompatible gel in a firstthree-dimensional device, contacting the first aliquot with a firstfluorophore dye selective for dead cells, the first fluorophore dyeemitting fluorescence at a first wavelength when bound to a dead cell,contacting the first aliquot with a second fluorophore dye selective forlive cells, the second fluorophore dye emitting fluorescence at a secondwavelength different from the first wavelength when bound to a livecell, measuring total fluorescence emitted by each of the first andsecond fluorophore dyes in the first aliquot, culturing the secondaliquot in a second three-dimensional device, contacting the secondaliquot with the first fluorophore dye, contacting the second aliquotwith the second fluorophore dye, wherein the contacting of the secondaliquot with the first fluorophore dye and second fluorophore dye iscarried out at least 24 hours after the contacting of the first aliquotwith the first fluorophore dye and second fluorophore dye, measuringtotal fluorescence emitted by each of the first and second fluorophoredyes in the second aliquot, wherein an increase or decrease in the ratioof live cells to dead cells in each of the aliquots may be assessed. 2.The method of claim 1, wherein the total fluorescence emitted by each ofthe first and second fluorophore dyes is measured using a camera,preferably at a resolution of at least 2×, at least 3×, at least 4× ormore, from directly above or below each three-dimensional device, andpreferably wherein the three-dimensional devices are place on a moveablestage permitting the camera to capture the total fluorescence in eachaliquot.
 3. The method of claim 1 or 2, wherein the second aliquot iscontacted with at least one test compound during the culturing of thesecond aliquot and wherein said culturing of the second aliquot in thepresence of the test compound occurs for at least 24 hours, at least twodays, at least three days, at least four days, at least five days, or atleast 6 days.
 4. The method of claim 1 or 2, wherein the second aliquotis contacted with at least two test compounds during the culturing ofthe second aliquot and wherein said culturing of the second aliquot inthe presence of the test compounds occurs for at least 24 hours, atleast two days, at least three days, at least four days, at least fivedays, or at least 6 days, and preferably wherein at least one of thetest compounds is an immune checkpoint inhibitor.
 5. The method of anyone of claims 1-4, wherein the first and the second aliquots eachcontain between about 15 and 30 spheroids, preferably between about 20and 25 spheroids.
 6. The method of any one of claims 1-5, wherein thefirst three-dimensional device is a first three-dimensional microfluidicdevice and the second three-dimensional device is a secondthree-dimensional microfluidic device, and optionally culturing thefirst aliquot in the first three-dimensional microfluidic device, saidculturing being for less than 6 hours, less than 3 hours, less than 2hours and even less than 1 hour prior to contacting the first aliquotwith the first and second fluorophore dyes.
 7. The method of claim 6,wherein the first aliquot in the first three-dimensional microfluidicdevice is not cultured prior to contacting the first aliquot with thefirst and second fluorophore dyes.
 8. The method of any one of claim1-7, wherein the enzyme is collagenase.
 9. The method of any one ofclaims 1-8, wherein the first fluorophore dye is propidium iodide,DRAQ7, 7-AAD, eBioscience Fixable Viability Dye eFluor® 455UV,eBioscience Fixable Viability Dye eFluor® 450, eBioscience FixableViability Dye eFluor® 506, eBioscience Fixable Viability Dye eFluor®520, eBioscience Fixable Viability Dye eFluor®660, eBioscience FixableViability Dye eFluor® 780, BioLegend Zombie Aqua™, BioLegend ZombieNIR™, BioLegend Zombie Red™, BioLegend Zombie Violet™, BioLegend ZombieUV™, or BioLegend Zombie Yellow™, and/or the second fluorophore dye isacridine orange, nuclear green LCS1 (ab138904), DRAQ5 (ab108410), CyTRAKOrange, NUCLEAR-ID Red DNA stain (ENZ-52406), SiR700-DNA, calcein AM,calcein violet AM, calcein blue AM, Vybrant® DyeCycle™ Violet, Vybrant®DyeCycle™ Green, Vybrant® DyeCycle™ Orange, or Vybrant® DyeCycle™ Ruby.10. The method of any one of claims 1-9, wherein the tumor spheroids areobtained by mincing a primary tumor sample in a medium supplemented withserum; treating the minced primary tumor sample with an enzyme; andharvesting tumor spheroids from the enzyme treated sample, andpreferably wherein the minced primary tumor sample is treated with theenzyme in an amount or for a time sufficient to yield a partialdigestion of the minced primary tumor sample, and preferably wherein thetreatment is for between 10 minutes and 60 minutes, and more preferablybetween 15 minutes and 45 minutes at a temperature of 25° C. to 39° C.11. The method of any one of claims 1-10, wherein the biocompatible gelis collagen, BD Matrigel™ Matrix Basement Membrane, or fibrin hydrogel.12. The method of any one of claims 1-11, wherein the tumor sample isderived from murine tissue of a Bladder MBT-2, Breast 4T1, EMT6, Colon,Colon26, CT-26, MC38, Fibrosarcoma WEHI-164, Kidney Renca, LeukemiaC1498, L1210, Liver H22, KLN205, LL/2, LewisLung, Lymphoma A20 S,E.G7-OVA, EL4, Mastocytoma P815, Melanoma B16-B16, B16-F10, S91, MyelomaMPC-11, Neuroblastoma Neuro-2a, Ovarian: ID8, Pancreatic PanO2,Plasmacytoma J558, or Prostate RM-1 murine model.
 13. The method of anyone of claims 1-11, wherein the tumor sample is a patient derivedxenograft (PDX).
 14. The method of any one of claims 1-13, wherein thethree-dimensional device comprises: one or more fluid channels flankedby one or more gel cage regions, wherein the one or more gel cageregions comprises the biocompatible gel in which the tumor spheroids areembedded, and wherein the device recapitulates in vivo tumormicroenvironment.
 15. The method of any one of claims 1-14, wherein thethree-dimensional device comprises: a substrate comprised of anoptically transparent material and further comprising i) one or morefluid channels; ii) one or more fluid channel inlets; iii) one or morefluid channel outlets; iv) one or more gel cage regions; and v) aplurality of posts; wherein all or a portion of each gel cage region isflanked by all or a portion of one or more fluid channels, therebycreating one or more gel cage region-fluid channel interface regions:each gel cage region comprises at least one row of posts which forms thegel cage region; and the one or more gel cage region has a height ofless than 500 μm.
 16. The method of any one of claims 3-15, wherein thefirst test compound is a small molecule, a nucleic acid molecule, anRNAi compound, an aptamer, a protein or a peptide, an antibody orantigen-binding antibody fragment, a ligand or receptor-binding protein,a gene therapy vector, or a combination thereof.
 17. The method of anyone of claims 3-15, wherein the first test compound is achemotherapeutic compound, an immunomodulatory compound, or radiation.18. The method of claim 17, wherein the first test compound is analkylating compound, an antimetabolite, an antrhracycline, a proteasomeinhibitor, or an mTOR inhibitor.
 19. The method of any one of claims3-15, wherein the first test compound is an immune modulator.
 20. Themethod of any of the preceding claims, further comprising isolating RNAfrom the first aliquot and second aliquot of tumor spheroids; andanalyzing gene expression of the first aliquot and second aliquot oftumor spheroids based on the isolated RNA, wherein the gene expressionis analyzed by performing RNA sequencing (RNA-seq) on the isolated RNA21. A method for detecting a change in tumor cell spheroids uponexposure to a test compound comprising: culturing a first aliquot oftumor cell spheroids in a first three-dimensional device; culturing asecond aliquot of tumor cell spheroids in the presence of a first testcompound in a second three-dimensional device; and detecting a change inthe second aliquot as compared to the first aliquot, wherein the changeis selected from: a clustering of immune cells around one or more of thetumor cell spheroids of the first or second aliquot; a decrease in sizeand/or number of the tumor cell spheroids of the first or secondaliquot; or a chemical change.
 22. The method of claim 21, wherein thesecond aliquot is cultured in the presence of the first test compoundand a second test compound.
 23. The method of claim 22, wherein thefirst aliquot is cultured in the presence of the first test compound.24. The method of any of claims 21-23, further comprising: culturing athird aliquot of tumor cell spheroids in the presence of the first testcompound and the second test compound in a third three-dimensionaldevice; and detecting a change in the third aliquot relative to thefirst and/or second aliquot.
 25. The method of any of claims 21-24,wherein the first aliquot is cultured in the presence of the secondand/or a third test compound.
 26. The method of any of claims 21-25,wherein the second aliquot is cultured in the presence of the thirdand/or a fourth test compound.
 27. The method of any of claims 21-26,wherein the third aliquot is cultured in the presence of the thirdand/or fourth test compound.
 28. The method of any of claims 21-27,wherein detecting a chemical change comprises detecting a presence of abiological molecule secreted into tumor cell spheroid cell culturesupernatant of the first, second, and/or third aliquots.
 29. The methodof claim 28, wherein the biological molecule is a protein, acarbohydrate, a lipid, a nucleic acid, a metabolite, or a combinationthereof.
 30. The method of claim 28, wherein the biological molecule isa chemokine or a cytokine.
 31. The method of claim 30, wherein thecytokine is a growth factor.
 32. The method of claim 28, wherein thebiological molecule is known to be associated with activation of theimmune system or otherwise an enhancement of the immune response. 33.The method of any of claims 21-27, wherein detecting a chemical changecomprises detecting a change in nucleic acid content.
 34. The method ofclaim 33, wherein detecting the change in nucleic acid content comprisesdetecting a change in extracellular nucleic acids.
 35. The method ofclaim 33, wherein detecting the change in nucleic acid content comprisesdetecting a change in nucleic acids isolated from tumor cell spheroidsfrom the first, second, and/or third aliquots.
 36. The method of claim34 or 35, wherein detecting the change in nucleic acid content comprisesdetecting a change in gene expression.
 37. The method of claim 36,wherein detecting a change in gene expression comprises detecting achange expression of genes associated with cytotoxicity.
 38. The methodof claim 37, wherein genes associated with cytotoxicity comprisecytokines and cytokine receptors.
 39. The method of claim 38, whereinthe cytokines comprise growth factors.
 40. The method of any of claims34-39, wherein detecting the change in nucleic acid content comprisesanalyzing DNA and/or RNA from the first, second, and/or third aliquotsof tumor cell spheroids.
 41. The method of claim 40, wherein RNA fromthe first, second, and/or third aliquots of tumor cell spheroids isanalyzed by RNA sequencing.
 42. The method of any of claims 21-41,wherein the first, second, third, and/or fourth test compound is a smallmolecule, a nucleic acid molecule, an RNAi compound, an aptamer, aprotein or a peptide, an antibody or antigen-binding antibody fragment,a ligand or receptor-binding protein, a gene therapy vector, or acombination thereof.
 43. The method of any one of claims 21-41, whereinthe first, second, third, and/or fourth test compound is an immunemodulator.
 44. The method of claim 43, wherein the immune modulatorcomprises immune activating compounds or inhibitors of an immunecheckpoint protein selected from the group consisting of CTLA-4, PD-1,PD-L1, TIM3, LAG3, B7-H3 (CD276), B7-H4, 4-1BB (CD137), OX40, ICOS,CD27, CD28, PD-L2, CD80, CD86, B7RP1, HVEM, BTLA, CD137L, OX40L, CD70,CD40, CD40L, GAL9, A2aR, and VISTA.
 45. The method of claim 43 or 44,wherein the immune checkpoint inhibitor inhibits PD1.
 46. The method ofany of claims 21-45, wherein the first test compound is an immunecheckpoint inhibitor and the second test compound is a small moleculecompound.
 47. The method of claim 46, wherein the small moleculecompound is a TBK-1 inhibitor.
 48. The method of any of claims 21-45,wherein the first, second, third, and/or fourth test compound is achemical from a test compound library.
 49. The method of claim 48,wherein the first test compound is an immune checkpoint inhibitor andthe second, third, and/or fourth test compound is a chemical from a testcompound library.
 50. The method of any of claims 21-49, wherein thefirst, second, and/or third aliquots are cultured in a biocompatiblegel.
 51. The method of claim 50, wherein the first, second, and/or thirdaliquots are suspended in a biocompatible gel in a fluid channel of thethree-dimensional microfluidic device before culturing.
 52. The methodof any of claims 21-51, wherein the first, second, and/or third aliquotsare obtained from an enzyme treated tumor sample.
 53. A method forevaluating tumor cell spheroids in a three-dimensional microfluidicdevice, the method comprising: culturing a first aliquot of tumorspheroids in a first three-dimensional device, culturing the secondaliquot of tumor spheroids in a second three-dimensional device in thepresence of a first test compound; isolating RNA from the first aliquotand second aliquot of tumor spheroids; and analyzing gene expression ofthe first aliquot and second aliquot of tumor spheroids based on theisolated RNA.
 54. The method of claim 53, wherein the second aliquot iscultured in the presence of a first test compound and a second testcompound.
 55. The method of claim 53 or 54, further comprising:culturing the third aliquot of tumor spheroids in a thirdthree-dimensional device in the presence of the first test compound anda second test compound; isolating RNA from the third aliquot of tumorspheroids; and analyzing gene expression of the third aliquot of tumorspheroids based on the isolated RNA.
 56. The method of any of claims53-55, wherein the second aliquot is cultured in the presence of a thirdand/or fourth test compound.
 57. The method of any of claims 53-56,wherein the third aliquot is cultured in the presence of a third and/orfourth test compound.
 58. The method of any of claims 53-57, wherein theRNA is isolated from the supernatant or from the cell culture of thefirst aliquot and second aliquot of tumor spheroids.
 59. The method ofclaim 53, wherein the gene expression of the first aliquot and secondaliquot of tumor spheroids is analyzed by performing RNA sequencing(RNA-seq) on the isolated RNA
 60. The method of any of claims 53-59,wherein the tumor cell spheroids are obtained from an enzyme treatedtumor sample.
 61. The method of any of claims 53-60, wherein the first,second, third, and/or fourth test compound is a small molecule, anucleic acid molecule, an RNAi compound, an aptamer, a protein or apeptide, an antibody or antigen-binding antibody fragment, a ligand orreceptor-binding protein, a gene therapy vector, or a combinationthereof.
 62. The method of any one of claims 53-61, wherein the firsttest compound is an immune modulator.
 63. The method of claim 62,wherein the immune modulator comprises immune activating compounds orinhibitors of an immune checkpoint protein selected from the groupconsisting of CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3 (CD276), B7-H4,4-1BB (CD137), OX40, ICOS, CD27, CD28, PD-L2, CD80, CD86, B7RP1, HVEMBTLA, CD137L, OX40L, CD70, CD40, CD40L, GAL9, A2aR, and VISTA.
 64. Themethod of claim 63, wherein the immune checkpoint inhibitor inhibitsPD1.
 65. The method of any of claims 53-64, wherein the first testcompound is an immune checkpoint inhibitor and the second test compoundis a small molecule compound.
 66. The method of claim 65, wherein thesmall molecule compound is a TBK-1 inhibitor.
 67. The method of any ofclaims 53-66, wherein the first, second, third, and/or fourth testcompound is a chemical from a test compound library.
 68. The method ofany of claims 53-67, wherein the first test compound is an immunecheckpoint inhibitor and the second, third, and/or fourth test compoundis a chemical from a test compound library.
 69. The method of any ofclaims 53-68, wherein the first, second, and/or third aliquots arecultured in a biocompatible gel.
 70. The method of claim 69, wherein thefirst, second, and/or third aliquots are suspended in a biocompatiblegel in a fluid channel of the three-dimensional microfluidic devicebefore culturing.